REVIEW OF LITERATURE -...

Chapter - II

REVIEW OF LITERATURE

II. 1. Human Serum Albumin

Human serum albumin (HSA) is the most abundant protein in blood

plasma with one liter of plasma containing about 60% HSA. There is about 42 ±

3.5 g of HSA per liter of human blood. HSA is a non glycoprotein, one of the

few secreted proteins which lacks carbohydrate (Peters, 1985). Produced in the

liver, HSA is largely responsible for maintaining normal osmolarity in the

bloodstream but also functions as a carrier for numerous endogenous and

exogenous compounds such as fatty acids, hormones, toxic metabolites, bile

acids, amino acids, drugs, and metals (Furukawa, 2011). Besides being present in

the plasma, HSA is also found in tissues and body secretions, skins and lymph

cavities. Under normal physiological conditions HSA contributes 80% of the

colloid osmotic pressure that provides the driving force to retain fluid within

blood vessels (25–33 mmHg). A reduction in the concentration of HSA in the

blood circulation results in edema, ascites, and pleural effusion. In addition to the

osmotic function, HSA provides a high-capacity reservoir to stabilize the

concentration of free ligands (Kragh-Hansen, 1990). HSA also functions as a

major antioxidant in human body (Hiroshi et al., 2001). Clinically, it is being

used as a plasma expander. The administration of HSA is generally considered to

be the gold standard for treating severe hypoalbuminemia as a result of

conditions such as burns, nephritic syndrome, reduction in the synthesis of HSA

induced by chronic liver cirrhosis, and hemorrhagic shock (Kazuaki et al., 2012).

HSA is the most well studied plasma protein and an attractive macromolecular

carrier due to its availability in pure form and its biodegradability, non-toxicity

and non immunogenicity (Kratz, 2008).

16 Chapter II

II. 1. 1. Functional and physical properties of HSA

HSA has various in vivo functional and physical properties which have

been exploited in a number of biopharmaceutical applications (Mead et al.,

2007). Its amphiphilic properties makes it suitable as an additive to inhibit

adsorption of the active protein to the container via competitive adsorption

mechanisms. Its surface active characters enable it to fulfill the role of surfactant,

thereby preventing protein aggregation. In some instances, it stabilizes the

conformational structure of active molecule to maintain its bioactivity throughout

the product shelf-life. The presence of 17 disulphide linkage provides the protein

remarkable stability and in vivo half-life of 15-20 days. HSA also has a high glass

transition temperature, which in combination with its amphiphilic nature makes it

an ideal vehicle for cryopreservation. In vitro, the molecule’s stability is

increased and it remains in solution at room temperature to sustain the shelf-life

of biopharmaceutical products. During manufacture, HSA can withstand heating

to 60°C for 10 hrs to facilitate viral inactivation (Burnouf, 2007).

II. 1. 2. Pharmaceutical applications of HSA

HSA is one of the most widely used proteins in the pharmaceutical

industry (Mead et al., 2007). It is clinically in great demand as a replacement

fluid of plasma expander or as a compensator for blood loss. It is also used as a

clinical tool for the delivery of many biological products (Peters, 1985; Peters,

1996; Varshney et al., 2010). HSA is widely used clinically to treat serious burn

injuries, plasmaphoresis, hemorrhagic shock, hypoproteinemia, fetal

erythroblastosis, ascites caused by cirrhosis of the liver diuretic-resistant oedema

and for some groups of surgical patients (Hastings and Wolf, 1992; Goodey,

1993). The protein has been described as the major colloid that retains fluid in the

vascular system, acting as a tramp streamer by dithering. HSA transport a mixed

cargo of metabolites around various organs (Kragh- Hansen, 1990). Among these

metabolites, long chain fatty acids are quantitatively the most abundant, with the

normal loading being approximately two fatty acids per molecule of albumin

Review of Literature 17

(Petitpas et al., 2001). HSA also binds bilirubin (Petersen et al., 2000; Weisiger

et al., 2001), amino acids, numerous drugs (Ozer and Tacal, 2001) and heavy

metals and is implicated in the transfer of many ligands across organ-circulatory

interfaces such as liver, intestine, kidney and brain. The molecule has been

succinctly described as the protein that makes blood thicker than water. In

addition to the use of HSA alone, drugs that can be transported in conjunction

with HSA have been recently approved by many countries and provide beneficial

effects. Products like, Abraxane® (American Bioscience, Los Angeles, CA),

which is an albumin-paclitaxel nanoparticle with a mean particle size of 130 nm,

was approved for the treatment of metastatic breast cancer. Albumin and

paclitaxel, in the form of a complex, have several beneficial effects; Abraxane®

accumulates in tumors through the enhanced permeability and retention (EPR)

effect of solid tumors, but a further albumin transport pathway mediated by Gp60

located on the surface of endothelial cells appears to be responsible for the tumor

uptake and tumor distribution of Abraxane® and the subsequent release of

paclitaxel (Schnitzer et al., 1992; Tiruppathi et al., 1997; Wang et al., 2009).

Several drugs and agents that use an albumin as a drug carrier were also

approved, and there are new drugs based on albumin currently in the

development and in the clinical trial stages (Table II-1) (Morales et al., 2007;

Flisiak et al., 2009; Flisiak et al., 2010; Kim et al., 2011; Kazuaki et al., 2012).

Albuferon® (Human Genome Sciences, Rockville, Maryland), a fusion protein of

albumin and interferon-α-2b for the treatment of hepatitis C infections, is

expected to improve the treatment by allowing a monthly dosing schedule in

clinics (Nelson et al., 2010; Zeuzem et al., 2010). In addition to drug

development in the pharmaceutical industry, there is a growing interest in

exploring various pharmaceutical applications of albumin as a drug carrier in

academic research.

18 Chapter II

Table II-1 - Albumin-based drugs that have received market approval or are in clinical trial

Product Name Formation with Albumin Application Eff ect of Albumin

Levemir® (Novo nordisk, Princeton, NJ)

The albumin-binding derivative of human insulin

Type 1 and 2 diabetes

Increment of half-life (native insulin, 4–6 min; Lavemir® , 5–7 h)

Nanocoll® (Nycomed Amersham Sorin, Milan, Italy)

Tecnetium99m (99mTc) aggregated Albumin

γ-emitting radionuclide imaging agent

Increment of the accumulation in targeting area

Albures® (Sorin Biomedical, Irvine, CA)

,,

,,

,,

Pulmolite® (Du Pont Pharma, North Billerica, NA)

,,

,,

,,

Draximage® (draxlmage Inc., London, UK)

,,

,,

,,

Abraxane® Albumin–paclitaxel nanoparticle

Solid tumors Increment of accumulation and uptake in tumor

Albuferon™ Fusion protein of albumin and interferon-α-2b

Hepatitis C infection

Increased blood retention

Vasovist® (Schering AG, Berlin, Germany

Gadofosveset reversibly binds to human serum albumin.

Magnetic resonance contrast agent

Higher relaxivity and extended intravascular enhancement

MARS™ (Gambro, Lund, Sweden)

Dialysis solution containing human serum albumin across a high-flux permeable membrane.

Liver failure Increment of effectively removing albumin-bound endogenous or exogenous toxin.

Many researchers have used different approaches to develop novel

albumin-based drug carriers and these can be generally categorized into three

main modifications: (a) low-molecular-weight proteins fused with albumin

(Furukawa et al., 2011), (b) polymerization (Bae et al., 2012, Ozgur et al., 2012),

(c) surface modification (Lu et al., 2007; Hirata et al., 2010). The main objectives

of these modifications were to prolong blood retention or to enhance the delivery

of a drug to the target area, and their effectiveness and usefulness for various

reports. Among these modifications, albumin dimerization has great potential and

advantages for clinical applications as both a plasma expander and a drug carrier,


whereas other forms of albumin modifications could achieve only one of these two

clinical applications (Taguchi et al., 2012). Furthermore, several investigators have

clearly shown that increased concentrations of albumin dimers are present in the

circulating blood of patients with chronic renal disease as compared with age-

matched healthy individuals (Scorza and Minetti, 1998; Ogasawara et al., 2006), as

well as the concentration of products resulting from oxidative damage in the blood

(Mimic-Oka et al., 1999; Richard et al., 1991). These results imply that the

albumin dimer also has the potential for its use as a biomarker of oxidative stress

(Kazuaki et al., 2012). Many other novel uses for HSA in biological applications

have recently been explored, such as carrier of oxygen (Tsuchida et al., 2009),

nanodelivery of drugs (Cai et al., 2006), and fusion of peptides (Subramanian et

al., 2007).

Almost all of the HSA used therapeutically or non-therapeutically is

currently purified from donated human plasma or whole blood (pHSA). Although

pHSA is generally considered to be safe, the supply and purity of plasma itself is

threatened by known or other emerging pathogens.

II. 1. 3. Traditional HSA manufacture

HSA is currently used in greater volume than any other pharmaceutical

solution, with worldwide manufacture in the order of hundreds of tons annually

(Matejtschuk et al., 2000). Since 1940, it has been produced by fractionation of

plasma obtained from donors (Cohn, 1941). Although blood plasma represents

the richest source of HSA, there are problems associated with the purity of HSA

obtained from human donor blood, currently the most available source of blood

plasma. Donated blood plasma can carry viruses like Human Immunodeficiency

Virus (HIV), Hepatitis C Virus (HCV), Hepatitis A Virus (HAV), variant

Creutzfeldt-Jakob disease (vCJD) and West Nile virus. Such viruses must be

removed during the process of HSA recovery from donated plasma and, although

historically HSA has been a safe product, it is constantly subject to potential risks

20 Chapter II

of contamination. In addition, large–scale production of HSA aggravates the

limited source of human blood (EMEA,CHMP 2004).

These concerns have impelled the regulatory authorities worldwide for

creating a myriad of regulations to limit the use of plasma-derived materials with

the aim of minimizing transmission risks and necessitating a dedicated drive from

within the industry to develop substitute products and ever-more sophisticated

tools for the detection, clearance and removal of adventitious agents from serum-

derived products. Beyond purity, safety and other issues, consistence of supply is

another major issue in HSA production. The fractionation industry has not always

been capable of providing an adequate HSA supply and shortages have been

encountered, particularly when there are shortages of donated human blood

plasma. To address this issue, pharmaceutical companies have turned towards the

development of recombinant versions of HSA.

II. 2. Recombinant HSA (rHSA)

The shortcomings of therapeutic protein production from blood plasma

have inspired the production of recombinant human serum albumin (rHSA). In

1981, Lawn and co-workers (Genentch Company, America) for the first time

expressed rHSA in E. coli successfully and achieved the patent; however, the

expression level is milligram level without the capability of industrialization.

Recombumin® is a recombinant albumin produced and patented by Aventis (a US

pharmaceutical company) (Chuang et al., 2002).

Equivalent blood plasma products using DNA technologies with

recombinant therapeutic proteins offer several potential advantages over human

donor plasma. Most notably, because they are expressed in bacteria or animal

cells, recombinant proteins are (theoretically) 100 percent risk-free from the viral

contaminants that human plasma derived products may contain. It would be a

great advantage to be able to use genetic engineering to obtain rHSA in good

yield and at lower cost, with no danger of contamination by human pathogens.

For this reason, great efforts have been dedicated to the production of this protein


on a large scale by transgenic organisms. To date, expression of rHSA has been

reported in a number of expression systems from bacteria to mammalian cells as

listed in table II-2.

Table II-2: Recombinant HSA expression in different heterologous organisms

Organism Expression level

(Highest reported yet)

Reference

Escherichia coli 7 % of TBP** (Laltta et al., 1990)

Saccharomyces cervisiae 85 mg/l (Okabayashi et al., 1991; Sleep et al., 1990)

Pichia pastoris 270 mg/l (Ohtani et al., 1998)

Kluyvermyces lactis 1g/l (Fleer et al., 1991; Saliola et al., 1995)

Bacillus subtilis 0.135% of TSP (Saunders et al., 1987; Mao et al., 2012)

Solanum tuberosum 0.2% of TSP (Sijmons et al., 1990; Farran et al., 2002)

Niccotiana tobaccum 0.7% of TSP (Millan et al., 2003; Sun et al., 2011)

Hansenula polymorpha 24.6 g/l (Kang et al., 2001; Youn et al., 2010)

Hevea brasiliensis 33 µg/ml (Arokiaraj et al., 2002)

Transgenic mice 11.9 g/l (Barash et al., 1996; Shani et al., 1992, Wu et al., 2012)

Oryza sativa 10.58% of TSP (Huang et al., 2005; He et al., 2011)

* TSP-Total soluble protein, **TBP- Total bacterial protein

The rHSA has to be priced competitively to make any impact on the HSA

market. So the production costs must be kept to an absolute minimum. Therefore,

any production process involving the use of expensive media of the type used in

mammalian cell culture is effectively excluded from consideration. Another

constraint is the high number of disulfide bridges and the size of the protein (66.5

kDa), which in terms of heterologous expression considered to be large. Attempts

to produce large quantities of such proteins in an intracellular form in

recombinant organisms often result in the accumulation of the protein in an

insoluble denatured form in inclusion bodies. In such cases the disulfide bonds

are usually formed incorrectly or not at all.

22 Chapter II

II. 2. 1. rHSA producton - Current strategies

The global annual demand for HSA has increased significantly from

about 300 tons in 2006 to about 500 tons in 2011(He et al., 2011). The ever-

growing emergence of new therapies and applications based on HSA protein has

been a major driving force in the development of new, safe and large-scale,

platforms for rHSA production. To meet growing consumer demands, bioreactors

are becoming increasingly more important aspect for the production of large

amounts of recombinant proteins, particularly in cases where traditional sources

are limited due to safety, cost and/or availability. With the aim of establishing an

rHSA-producing organism for large scale industrial use, studies have been

performed on number of expression systems. The advances made in this aspect

have seen the development rHSA production in a number of expression system

based on a wide variety of living cells and organisms.

II. 2. 1a. Bacterial expression

The first expression system to produce rHSA was E. coli (Lawn et al.,

1981). The expression of heterologous genes in bacteria is the simplest and most

inexpensive way available for obtaining large amounts of desired proteins for

research, industrial and clinical research purposes. The E. coli expression system

has a proven track record since the first expression of somatostatin by Itakura and

co workers (1977), and is considered to be a milestone in genetic engineering.

Bacterial expression is the first expression system introduced in the biotech

industry (recombinant insulin) and gram negative bacteria is still used for

expression of a wide range of biopharmaceutical products.

Generally, there are three forms of foreign proteins expressed in E. coli

bacteria: (i) Fusion proteins are formed by fusing the gene of interest to other

sequences encoding a FLAG epitope tag, a poly-histidine tag, a fluorescent

protein, etc. This can facilitate further protein enrichment (e.g., by

immunoprecipitation), purification, or monitoring, and often affords protection

against degradation by intracellular proteases (Kim et al., 2002; Magliery et al.,


2005; Rosenfeld et al., 2005). (ii) Secreted proteins can accumulate in either the

periplasm or extracellular medium. This process can reduce potential degradation

by intracellular proteases, simplify the purification procedure, and contribute to

the correct formation of spatial structures in proteins secreted into the medium

(Stader and Silhavy, 1990; Makrides, 1996). Although some recombinant

proteins expressed in E. coli can be secreted into the periplasm, they cannot be

secreted into the extracellular medium in most circumstances. (iii) Inclusion

bodies are largely protected from proteolytic degradation by host cell enzymes.

However, the protein product may typically occur as an insoluble, misfolded

inclusion body, so subsequent solubilization and refolding steps arerequired

(Clark, 1998b; Marston, 1986).

rHSA has been expressed as insoluble aggregates in the cytoplasm of

E.coli (Lawn et al., 1981). However, there are distinct disadvantages in

producing HSA in E.coli. For example, conversion of the insoluble product to the

soluble form, which is frequently inefficient in vitro, is both time-consuming and

expensive. Besides, HSA derived from E.coli may be contaminated by endotoxin,

which causes an adverse reaction in patients (Okabayashi et al., 1991).

Saunders and his co-workers have studied the secretion of rHSA from

Bacillus subtilis using bacterial signal sequences (Saunders et al., 1987). The key

advantages of such a route are that the secreted protein is, by definition, soluble

and will not require disruption of the host organism prior to protein purification

(Goodey, 1993). One problem with rHSA accumulation that has been observed is

that a proportion of the secreted rHSA molecules are shorter than the full-length

protein, the nature and amount of these truncated molecules vary according to the

producer organism, growth regime leader sequence adopted (Sleep et al., 1990).

These molecules may be the result of proteolytic degradation of the full-length

protein or incomplete translation of the mRNA. In case of Bacillus subtilis,

however, incorrect processing of rHSA was reported and improvement by gene

manipulation resulted in only minimal secretion. It was found that the efficiency

24 Chapter II

of processing of the leader peptides is inversely correlated with the productivity.

For commercial production of rHSA at an economically feasible level, several

grams of rHSA secretion into the culture broth seemed to be the minimum

requirement for the starting material to be purified (Saunders et al., 1987).

The disadvantages of bacterial expression system include intra-cellular

expression in gram-negative bacteria (although some systems have been designed

for periplasmatic expression of the target protein). Lack of post-translational

modifications and protein folding mechanism limits hetrologus protein

expression.

II. 2. 1b. Yeast expression of rHSA

Currently, Saccharomyces cerevisiae, Kluyveromyces lactis, Hansenula

polymorph and Pichia pastoris (Sleep et al., 1990; Fleer et al., 1991; Kang et al.,

2001; Kobayashi et al., 2000) are the common yeast systems for heterologous

expression of rHSA. The yeast expression system offer high efficiency in terms

of short doubling times, high cell densities, high yields and low fermentation

costs. The production levels ranges from milligrams product per liter of culture to

several grams, depending on choice of yeast strain, type of product and culture

conditions. Yeast is easy to grow in large scale with simple nutritional demands

that lower the media cost. Yeast is given generally regarded as safe (GRAS)

status. In contrast to E. coli, yeast can express correctly folded proteins directly to

the medium, which greatly facilitates purification (low level of host cell proteins,

non-viscous solution, low level of DNA). The rigid cell wall renders the use of all

sorts of bioreactors possible regardless of stirring and shaking mechanisms.

S. cerevisiae has long been known to secrete few or no extracellular

proteases, however investigations carried out in recent years have begun to reveal

that extracellular proteolysis represents one of the most significant barriers to the

secretory production of heterologous proteins in this host organism. The main

problem in the production of rHSA in S. cerevisiae was found to be the

extracellular proteolytic cleavage of the recombinant protein during prolonged


culture (Kang et al., 2000). In another study, rHSA expressed in the yeast S.

cerevisiae accumulated in the insoluble fraction in a denatured form (Quirk et al.,

1989). Extraction of the product requires cell breakage, solubilization, reduction

and renaturation prior to purification (Goodey, 1993). Such downstream

processing would not be practical at the scale envisaged for rHSA and would

certainly not be cost effective. It was reported that the electrophoretic karyotype

of S. cerevisiae may not necessarily be stable. For example, during serial

cultivation of S. cerevisiae wild strain, modifications of electrophoretic karyotype

were observed at 55 generations (Longo and Vezinhet, 1993).

The methylotrophic yeast Pichia pastoris seemed an attractive candidate

as its system usually possesses a high production level. The high stability of

chromosomal DNA in large-scale fermentation is required for stable production,

especially for pharmaceutical products. From this point of view, the gene stability

of a P. pastoris recombinant strain expressing rHSA after long-term serial

cultivation under the conditions of vegetative and non- selective growth was

investigated (Ohi et al., 1998). Isolates from 83 generations showed HSA

production, suggesting that elimination of HSA expression constructs hardly

occurred during serial cultivation. In addition, the cells of each generation

produced the almost same amount of HSA, approximately 110 µg/ml. Thus, P.

pastoris was found to have superior gene stability; therefore, it is regarded as a

suitable host for rHSA gene expression even on an industrial scale.

Kluyveromyces lactis is another promising organism that can be used as

an alternative host due to its good secretory properties. K. lactis is aerobic yeast

which is able to grow on lactose as the sole carbon source. The conditions used

for cultivation of this organism on the large scale have been well established. K.

lactis has been successfully used as an alternative host to S. cerevisiae for

heterologous gene expression, and good transformation systems and stable

multicopy vectors are now available for the genetic manipulation of this yeast

(Fleer, 1992; Romanos et al., 1992). Under the control of the KlADH4 promoter

26 Chapter II

K. lactis cells grow in complex and defined media resulting in a high biomass

production (70 to 80 g dry-cell weight/l), and a significant amount of rHSA

production was observed in fed-batch cultures (1 g/l) when compared to batch

cultures (0.2 to 0.3 g/l) (Saliola et al., 1999).

Yeast cell cultures may be infected with bacteria and fungi. Filtration,

centrifugation and expanded bed technologies are used to harvest the cell culture.

Expanded bed technology combines separation of cells and product and capture

as the target protein binds to the suspended resin, while cells and culture medium

are passing through the column. If filtration or centrifugation is used, the

supernatant is applied directly to the capture column or after ultra-filtration with

the purpose of sample concentration. Hydrophobic interaction chromatography

should be avoided in the capture operation, as anti-foam agents added during

fermentation will bind to the column thus reducing the binding capacity. The

following purification and polishing steps are straight forward and yeast is

regarded as one of the most safe and simple expressions in use. As with E. coli,

this expression system is not able to produce post-translational modified proteins.

Viruses are of no concern with this expression system and relative low levels of

host cell proteins, nucleic acids and endotoxins (e.g. microbial contamination

during downstream operations) should be expected. But, problems are with

correct post-translational modifications, extensive proteolysis of target proteins

can occur. These restrictions limit the use of this expression system from

different heterologous proteins.

II. 2. 1c. Mammalian cells

Mammalian cell expression systems have several advantages over the

systems discussed above in that they promote signal synthesis, process, and can

secrete and glycosylate proteins, particularly eukaryotic proteins. Glycoproteins

are generally synthesized in mammalian cells because common microbial hosts

like E. coli lack the requisite machinery to synthesize appropriate glycoforms of

proteins. Several rodent or human derived cells like 3T3, CHO, BHK, HeLa and


HEpG2 are frequently used in biomedical research for hetrologus protein

expression. Despite the availability of a plenitude of cell lines, nearly 70% of all

recombinant protein therapeutics produced today is made in Chinese Hamster

Ovary (CHO) cells. The current annual sales for biological produced using CHO

cells alone exceed US $30 billion worldwide.

Mammalian cell expression systems can be used for either transient or

stable expression, depending upon the purpose of the expressed product. COS-

cell-based transient expression systems (COS TES) are commonly used for the

former and Chinese hamster ovary (CHO) cell stable expression systems (CHO

SES) for the latter (Edwards and Aruffo, 1993). CHO cells are used for rHSA

fusion protein production to improved half-life of precious proteins. Recombinant

human albumin-linker-erythropoietin (HSA-EPO) fusion protein was expressed

in CHO cells with EPO immunogenicity, which serve as foundation for the

development of long-lasting recombinant HSA-EPO protein (Joung et al., 2009;

Huang et al., 2011).

Since the introduction of continuous mammalian cell lines for the

production of recombinant biopharmaceuticals (e.g. interferon), this expression

system has been the second most used despite relatively high fermentation costs,

slow growth, relatively low yields and potential risk for viral infections. This

major achievement was made possible by a combination of technical progress

(bioreactors) and strict demands to cell line safety and characterization, use of

tested raw materials, good manufacturing practices, in process control and drug

substance/product quality control programs.

The relative low expression levels combined with high prices on culture

media and expensive quality control programs makes it generally more expensive

to produce recombinant proteins in animal cells than in microbial systems. This

system is capable of expression and secretion of complex posttranslational

modified proteins. The availability of good regulatory track record and

commercially available vectors are plus points of this expression system.

28 Chapter II

Mammalian cell-based bioreactors are, however, very expensive to develop and

maintain, and are plagued by complex nutrient requirements, poor oxygen and

nutrient distribution, waste accumulation, contamination by pathogens, and high

sensitivity of cells to shear stress (Wurm, 2004; Zhang et al., 2010).

Proteins produced in cell culture systems are generally purified from the

culture supernatant of lysed cells, an expensive process. Although the relatively

high cost, complicated technology, and potential contamination with animal

viruses of mammalian cell expression have been bottlenecks for its use in large-

scale industrial production, this system is often utilized to express many

heterologous proteins including viral structural protein and bioactive peptide for

specific functional analysis (Zimmer et al., 2003; Nagpal et al., 2004; Ren et al.,

2006).

II. 2. 1d. Transgenic animals

Since Gordon and team (1987) first reported that plasminogen could be

produced in mammary glands of the lactating mouse by transgenic technology,

there has almost been no arguement for the advantages and possibilities when

using transgenic animal bioreactors to generate the valuable pharmaceuticals.

Even complex post-translational modified proteins are successfully expressed in

their native biologically active form, thus making it possible to produce plasma

proteins, human antibodies and other proteins not easily derived from other

sources at industrial scale. It takes 18-33 months from introduction of gene to

production at usable levels. The animal husbandry and milking procedures are

known technologies upgraded to Good Agricultural Practices (GAP). The target

protein is usually expressed in the mammary gland, often at high protein

concentrations (5-50 g/l milk) far exceeding other expression systems.

Human serum albumin was first expressed in mammary glands of

transgenic mice (Huang et al., 2001). Though earlier study by Barsh and team in

1996 gave a suppression in hALB expression (Barsh et al., 1996), the results of

Ying and co-workers (2001) were positive and high level of hALB expression


was reported. The harvest procedure used for cell-based cultures is replaced by

fat and casein removal operations known from the dairy industry. Recently a high

level expression of HSA in milk from transgenic mice was reported (Wu et al.,

2012)

In comparison, the cost of producing 100 kg of protein from transgenic

goats’ milk has been estimated at $105/gram and manufacture using mammalian

cell culture costs $300-3000/gram depending on the product yield and other

operating factors (Young et al., 1997). Two adventitious agents are of concern:

viruses and prions; the latter have never been observed in milk and for that reason

the safety risk is expected low. Never the less, the safety of transgenic animal

derived products has been continuously challenged and is perhaps the main

reason why this expression system has not found widespread usage. The

downstream process very much resembles that of mammalian cell expression

systems including virus inactivation and filtration steps. Relative long period

from introduction of gene to production at usable levels and co-expression of

animal protein is also a major concern with this expression system.

II. 2. 1e. Plant-based bioreactors

The use of plants and plant cell suspension cultures is a relatively new

strategy for the production of recombinant proteins and has been adopted to

express many important pharmaceutical proteins (Brandsma et al., 2010; Shih

and Doran, 2009). Protein production in plants provides a number of advantages

not found in other production platforms. First, a major advantage that all plant

protein production systems have over cell culture systems (including bacteria,

yeast, and mammalian cell culture) is the potential for significant reduction in

cost. It is estimated that protein production in transgenic plants can be as much as

four orders of magnitude less expensive than production in mammalian cell

culture, on a per gram of unpurified protein basis (Dove, 2002). Secondly, plant-

produced proteins are not susceptible to viral or prion contamination that can

harm humans, as is always a concern with animal cell culture (Chebolu and

30 Chapter II

Daniell, 2009). Third, as eukaryotes plants possess the chaperones and cellular

machinery required to fold complex human proteins that bacteria and yeast may

not be able to process properly (Franklin and Mayfield, 2004). Finally, most

species of plants are considered GRAS (generally regarded as safe) (Rosenberg et

al., 2008), meaning that if the protein can be expressed in a bioavailable form;

purification steps could potentially be eliminated altogether.

Studies have shown that rHSA can be produced in leaves of transgenic

tobacco and potato and tobacco suspension cells (Sijmons et al., 1990). The

transgenic constructs contained either the authentic HSA signal peptide or a

preseqeunce from the extracellular PR-S protein from tobacco fused to the coding

region of HSA. The secreted protein was either isolated from leaf tissue or from

plant cell suspension cultures. Chromatographically purified rHSA was analysed

by N-terminal amino acid sequencing. The recombinant protein containing the

human signal peptide was not correctly processed, while the construct with the

plant specific prepropeptide allowed the production of correctly cleaved rHSA in

transgenic plants, and transgenic tobacco suspension cell lines. Plant specific

signal sequence facilitates secretion of 0.25µg rHSA/mg protein into the culture

medium. However, expression levels were, too low for most commercial

applications being 0.02% of the total soluble protein in shoot and young leaves,

and five times lower in roots and tubers.

Farran and his co-workers studied expression of rHSA in tubers of

transgenic potato plants (Farran et al., 2002). HSA gene was cloned under the

B33 promoter of patatin (a specific tuber promoter). The rHSA amino terminal

was fused to a tuber signal peptide (protease inhibitor II) and Agrobacterium

mediated gene transfer to potato leaves was performed. rHSA was detected in the

tubers of transgenic potato plants, the highest accumulation level reaching to

0.2% of the tuber soluble protein. This higher level than the previous report

(Sijmons et al., 1990) was attributed to the differences in the expression cassettes

used for transformation.


Ideally, in order for the bioreactor system to be commercially viable, the

expression levels should be in excess of 5% TSP. The expression levels of

recombinant proteins in transgenic plants are highly variable and range from

levels in the order of 0.01% (Mason et al., 1992) to 46.1% (De Cosa et al., 2001)

of total soluble protein (TSP). Studies have shown that the cost of producing

recombinant proteins in plants could be 10-50-fold lower than producing the

same protein by E. coli fermentation, depending on the crop, and that this gap

will widen as production reaches agricultural scale (Kusnadi et al., 1997). A

recent study in Oryza sativa has come up with high level of rHSA production,

10.58% of TSP (He et al., 2011) which is found to be a promising result for

commercial production.

Generally plants are not hosts to major pathogens that can contaminate

bacterial and mammalian systems (Herbers and Sonnewald, 1999). There is

however at least one pathogen, Pseudomonas aeruginosa, that can cause disease

in both plants and animals (Rhame et al., 1997), and some toxin-producing

bacteria and fungi occur in plants. To date, plant viruses are not known to infect

humans or animals. Potentially pathogenic human and animal viruses are not

capable of replicating in plant cells (Miele, 1997). There are, however, potential

concerns that must be addressed. These include: containment of genetically-

modified plants in the environment (gene flow); allergic reactions to plant protein

glycans and other plant antigens; product contamination by mycotoxins,

pesticides, herbicides and endogenous plant secondary metabolites and regulatory

uncertainty for proteins requiring approval for human drug use.

Although several systems for the expression of rHSA have been tested

and found to be successful, none is yet commercially viable (Desai et al., 2010).

This knowledge has lead research community to search for newer, more efficient

and economical expression systems. Microalgae are expected to be ideal

candidates for this requirement.

32 Chapter II

II. 3. Microalgae

Unicellular green microalgae are of interest in biological research as

simple models for higher plants and from a biotechnological perspective as a

natural source of high value compounds for use in health and medical

applications. The current world production of raw microalgal biomass exceeds

5000 tons, generating an estimated $1–1.25 billion in revenue every year (Pulz

and Gross, 2004; Walker et al., 2005a; Spolaore et al., 2006). Fundamental

research has focused on the alga Chlamydomonas reinhardtii as a model plant

system for the study of photosynthetic and other metabolic pathways, the

biogenesis of subcellular components and cell-cycle control. On the other hand,

species of Dunaliella, Haematococcus, Chlorella and Euglena produce

carotenoids such as β-carotene, astaxanthin and canthaxanthin used as pigments

in food products and cosmetics, vitamin A antioxidant supplements, health food

products, and feed additives for poultry, livestock, fish and crustaceans

(Borowitzka, 1988; Lorenz and Cysewski, 2000). For example, over 80% of the

world’s supply of natural β-carotene comes from the halophilic green alga

Dunaliella salina harvested from large saline ponds (Curtain, 2000).

Microalgae are still mainly used as nutritional supplements for human and

animal consumption due to their high protein and vitamin contents. They have,

however, also elicited interest as production systems for a host of valuable

naturally-produced compounds (Harun et al., 2010). The most notable of these

applications is the cultivation of the green algae Haematococcus pluvialis for its

high-level production scaling up to 50 mg/g dry cell weight (DCW) of

astaxanthin, a carotenoid pigment widely used as a feed additive in fish

aquaculture, and prized by the pharmaceutical and cosmetic industries for its

antioxidant properties (Hyunsuk et al., 2005; Kathiresan and Sarada, 2009).

Microalgae could potentially be a future commercial source of a number

of other vitamins including vitamin C, E and B12 (Borowitzka, 1988).

Pharmacologically active compounds have also been reported in microalgae,


including compounds with anticancer, antimicrobial, antiviral, and various

neurological activities (Schwartz et al., 1990; Cannell, 1993; Codd, 1995; Moore,

1996) as well as lipids that are rich in long-chain polyunsaturated acids of interest

in the treatment of heart disease (Simons et al., 1985) and as anti-inflammatory

agents (Lee et al., 1985).

Given their relatively high oil content, microalgae are of considerable

interest for the biodiesel industry (Williams and Laurens, 2010). Although

microalgal oil contents as high as 75% DCW have been reported, such contents

are highly unusual and obtained only under of specific cultivation conditions, and

therefore do not apply to current industrial systems. The microalgal systems used

in practical applications typically have oil contents closer to 20– 40% DCW. It

has been argued that microalgae may present the only economically and

environmentally viable feedstock for biofuel production. Estimates based on oil

contents of 70% DCW, which some believe to eventually be reachable as

research progresses, place the cost of microalgae-derived biodiesel at 0.72 USD/l,

which currently exceeds the viability of alternate biofuel feedstocks (Chisti,

2007). Considerable progress has been achieved in regards to increasing the oil

yield of microalgae (Courchesne et al., 2009), and several reviews on

microalgae-based biodiesel production have recently been published (Sialve et

al., 2009; Brennan and Owende, 2010; Greenwell et al., 2010; Mata et al., 2010;

Smith et al., 2010; Williams and Laurens, 2010). Several reviews are available on

microalgae based cultivation methods (Carvalho et al., 2006; Eriksen, 2008 and

Xu et al., 2009).

Microalgae have been used in health food and cosmetic industry for a

long time as they represent a natural source of lipids, vitamins, pigments and

antioxidants (Potvin and Zhang, 2010). The harvesting of products from

microalgal culture has a number of advantages from an industry perspective,

which has led to an increased interest in the biotechnology of this group of

organisms. Microalgae are photoautotrophic obviating the need for an exogenous

34 Chapter II

carbon source for energy and making their large-scale culture comparatively

cheap. Many microalgae grow in saline to hyper-saline waters and thus their

large-scale culture does not compete with conventional agriculture for the limited

resources of arable land and fresh water. Furthermore, microalgae such as

Dunaliella, Haematococcus and Chlorella which do not produce toxins and are

classified as food sources. Naturally, the potential for large-scale culture in

conditions unsuitable for conventional crops makes microalgae a desirable target

for both increased production of natural compounds by metabolic engineering

and for exploitation as biological factories for the synthesis of novel high-value

compounds. While the utility of Chlamydomonas as a model organism has been

greatly facilitated by the development of genetic transformation procedures

(Stevens and Purton, 1997), the lack of efficient genetic transformation systems

for other microalgal species has been a major limitation in the manipulation of

these organisms, and current strategies for increasing product yield rely on

standard techniques such as mutagenesis and selection, or trial and error testing

of growth conditions.

The development of systems for algal transformation system has

advanced significantly in recent years; success with Chlamydomonas has led to

the development of transformation systems for related algae, most notably Volvox

carteri, and several diatom species. Success in this area has required novel

approaches as most algal groups have shown significant natural resistance to

many of the antibiotics and herbicides commonly used for selection (Apt et al.,

1996) also the high salt conditions required for the growth of many marine algae

may reduce the activities of antibiotics (Allnutt et al., 2000).

II. 3. 1. Microalgae: as an expression system

In addition to their value as platforms for the production of naturally-

produced compounds, microalgae have been eliciting considerable interest over

the last decade as recombinant protein expression systems, as they combine the

rapid growth and ease of cultivation inherent to many microorganisms with the


ability of plant cells to perform post-transcriptional and post-translational

modifications. Research into transgenic microalgae is fuelled by the worldwide

demand for recombinant proteins and other bio products, the market for which is

growing exponentially and was expected to be $70 billion in 2010 (Pavlou and

Reichert, 2004).

Algae possess a number of advantages over transgenic plant systems for

the production of recombinant proteins. They can be grown in contained

bioreactors, reducing the risk of contamination of the production system by

airborne contaminants, and also protecting the environment from any potential

flow of transgenes into the surrounding ecosystem. Growth in containment also

greatly reduces the potential for loss of the crop due to predation or pathogen

attack. Algae progress from initial transformation to large-scale protein

production in a matter of weeks, compared to timescales on the order of months

or years in higher plants such as corn or tobacco (Franklin and Mayfield, 2004).

As micro-algae are all a single cell type, there should also be less variation in

recombinant protein accumulation, making downstream processing more

uniform.

II. 3. 2. Advantages of therapeutic protein production in microalgae

Although transgenic microalgal technology is still in its infancy,

microalgae may represent the ‘best of both worlds’ by combining simple and

inexpensive growth requirements and capabilities for post-transcriptional and

post-translational processing of plants, with the rapid growth rate and potential

for high-density culture of microorganisms (Walker et al., 2005a). Unicellular

photosynthetic green algae are most commonly used for protein production as

they only require inexpensive salt-based media, carbon dioxide and light for

growth. Most green algae are also classified as generally regarded as safe

(GRAS), making purification and processing of expressed products much less

onerous for many targeted applications. Contrary to transgenic plants which must

be strictly contained to avoid the transfer of transgenic material to surrounding

36 Chapter II

wild-type flora by airborne vectors, microalgae can be cultivated in open

facilities as no such transfer can occurs. On the economics side, based on

recombinant antibody production studies, the cost of production per gram of

functional antibody is $150, $0.05 and $0.002 in mammalian, plant and

microalgal bioreactor systems respectively, making the latter system very

economically attractive (Mayfield et al., 2003). A comparison of different

recombinant protein expression systems is shown in table II-3.

Table II-3: Comparison of different recombinant protein expression systems.

Expression Systems

Expression system Characteristics

Molecular Operational

Glycosylation Gene size Sensitivity to shear stress

Recombinant Product yield

Production time

Cost of cultivation

Scale up

cost

Cost of storage

Bacteria None Unknown Medium Medium Short Medium High Low (-20°C)

Yeast Incorrect Unknown Medium High Medium Medium High Low (-20°C)

Insect

Correct, but depends on strain and product

Limited High Medium to

High Long High High

High (Liquid N2)

Mammalian Cells

Correct Limited High Medium to

High Long High High

High (Liquid N2)

Plant Cells Correct Unlimited N/A High Long Low Very low

Low (Room temperature)

Unicellular Microalgae Correct Unlimited Low Generally low Short Very low Low

Low (Room temperature)

Despite the recent surge of interest and successful transformation of a myriad

of microalgal species, transgenic strains belonging to the Chlamydomonas,

Chlorella, Volvox, Haematococcus and Dunaliella genera remain the most widely

used and studied (Griesbeck et al., 2006; Raja et al., 2008; Rosenberg et al., 2008),

and many obstacles remain to be overcome before microalgae can be considered

standard expression systems. The large majority of current work is performed with

Chlamydomonas reinhardtii, as it is the best characterized of the microalgal species.

Considerable progress has been made in metabolic engineering towards increasing

the expression of naturally produced compounds, with varying levels of success


(Rosenberg et al., 2008). Although recombinant protein production is notably

hindered by low expression levels, the continuing development of genetic

engineering tools for microalgae has allowed the expression of fully functional

antibodies (Franklin and Mayfield, 2005; Tran et al., 2009), therapeutics (Boehm,

2007; Weathers et al., 2010), and bactericides (Li and Tsai, 2009) at economically

viable levels. Despite this progress, however, success essentially remains anecdotal

and no wide-ranging system or protocol leading to high-level expression has been

established till date. The recent advances made in C. reinhardtii makes this

microalga an ideal candidate for future studies.

II. 4. Chlamydomonas reinhardtii

Chlamydomonas is a unicellular, freshwater dwelling green microalga.

Highly adaptable, these green algae live in many different environments throughout

the world. Chlamydomonas can grow on a simple medium of inorganic salts in the

light, using photosynthesis to provide energy. They can also grow in total darkness if

acetate is provided as a carbon source for chemosynthesis. Chlamydomonas is the

model organism for physiological, biochemical, and molecular studies of

photosynthesis and flagella function (Harris, 2001), Chlamydomonas reinhardtii

over the years emerged as the predominant laboratory species of Chlamydomonas,

primarily owing to its ability to grow non-photosynthetically with acetate as its sole

carbon source.

The wild-type C. reinhardtii cell averages about 10 mm in diameter (with

significant variation through the cell cycle) and is enclosed within a wall consisting

primarily of hydroxyproline-rich glycoproteins that resemble plant extensins.

Contrary to a few erroneous early reports that have unfortunately been perpetuated in

some textbooks, the C. reinhardtii cell wall does not contain cellulose. The wild-type

wall comprises seven principal layers. Genes for some wall components have been

cloned and sequenced, and many mutants with defects in cell wall biogenesis have

been isolated. Cell wall mutants have found widespread use as recipients for

transformation with exogenous DNA, a process that is much more efficient with

38 Chapter II

wall-less cells. Vegetative cells are normally haploid, but stable diploids can be

selected using auxotrophic markers.

The 15,758 bp mitochondrial genome is linear and contains only a few

genes: cob, cox1, five subunits of mitochondrial NADH dehydrogenase, the

mitochondrial rRNAs (which are fragmented in the DNA sequence); three tRNAs,

and an opening reading frame that resembles a reverse transcriptase (GenBank #

NC001638) (Vahrenholz et al., 1993). In 2002, the complete sequence of the

Chlamydomonas chloroplast genome was released (GenBank #BK000554) which

consists of 203,395 bp (Maul et al., 2002). DOE Joint Genome Institute in 2007

completed the whole genome sequencing of Chlamydomonas. The nuclear genome

size is estimated to be 121-megabase (Mb), generated by whole-genome, shotgun

end sequencing of plasmid and fosmid libraries, followed by assembly into ~1500

scaffolds. The Chlamydomonas nuclear genome comprises 17 linkage groups

presumably corresponding to 17 chromosomes (Merchant et al., 2007).

Chlamydomonas is also highly amenable to genetic analysis with

transformation systems for all three genomes (nuclear, chloroplast and

mitochondrial). The complete characterization of the genomes of the mitochondrion

and the chloroplast along with the recent completion of the nuclear genome sequence

provides unique opportunities in functional genomics and will further add to the

value of this organism as a model system. For the selection of an ideal expression

system, major requirements are the availability of various selectable markers,

reporter genes and promoters. In C. reinhardtii a lot of studies have been carried out

in this regard successfully. In addition to that, a vast number of methods for genetic

manipulation have been developed for C. reinhardtii over the last few years making

it a top candidate for biotechnological applications (Fuhrmann et al., 2002).

II. 4. 1. Therapeutic protein production in Chlamydomonas reinhardtii

Expressing therapeutic proteins in micro-alga C. reinhardtii has several

advantages over other systems employed today. The length of time required from the

initial transformation event to having usable (milligram to gram) quantities of a


protein can be relatively quick in algae. Stable transgenic lines can be generated in as

little at 10 days, and scale-up to production volumes can potentially be achieved a

few weeks after this. In addition, algae can easily be grown in full containment

reducing any concern about environmental contamination of the therapeutic protein.

Moreover, because algae do not have pollen, there is no potential for the introduction

of transgenes into food crops, as potentially could occur in higher plants by gene

flow (via pollen) to surrounding plants (Mayfield et al., 2007). Finally, many green

algae fall into the category of Generally Recognized as Safe (GRAS), meaning they

are safe to eat, and are therefore potentially a source for the oral delivery of

therapeutic proteins, perhaps with little or no purification.

There is a growing interest in exploiting Chlamydomonas for

biotechnological purposes, for the contained and cost-effective expression of

biopharmaceuticals, an area commonly referred to as molecular farming (Franklin

and Mayfield, 2004; Walker et al., 2005a). Several transgenes with a pharmaceutical

or biotechnological background have been successfully expressed in C. reinhardtii

(Table II-4) (modified from Specht et al., 2010). They can be grouped into proteins

involved in metal metabolism, antigenic proteins, and antibodies. Whole

Chlamydomonas cells could be used as edible vaccines in industrial farming

processes superseding the purification of the immunogen. It has been suggested that

an oral application of such modified algal strains in food or drinking water can also

stimulate antibody production in mammals.

40 Chapter II

Table II-4: Recent successes in therapeutic protein production in Chlamydomonas reinhardtii

Gene expressed

Function Expression

level Source

HSV8-lsc First mammalian protein expressed, antibody

Detectable (Mayfield et al., 2003)

CTB-VP1 Cholera toxin B subunit fused to foot and mouth disease VP1

3% TSP (Sun et al., 2003)

HSV8-scFv Classic single-chain antibody 0.5% TSP (Mayfield et al., 2005)

hMT-2 Human metallothionine-2 Detectable (Zhang et al., 2006)

hTRAIL Human tumor necrosis factor-related apoptosis-inducing ligand

~0.67% TSP (Yang et al., 2006)

M-SAA Bovine mammary-associated serum amyloid

~5% TSP Manuell et al., (2007)

CSFV-E2 Swine fever virus E2 viral protein ~2% TSP (He et al., 2007)

hGAD65 Diabetes-associated anutoantigen human glutamic acid decarboxylase 65

~0.3% TSP (Wang et al., 2008)

ARS2-crEpo Human erythropoietin fused to ARS2 export sequence

100 lg/l culture (Eichler-Stahlberg et

al., 2009)

83K7C Full-length IgG1 human monoclonal antibody against anthrax protective antigen 83

0.01% dry algal biomass

(Tran et al., 2009)

IgG1 Murine and human antibodies (LC and HC) Detectable (Tran et al., 2009)

VP28 White spot syndrome virus protein 28 ~10.5% TSP (Surzycki et al., 2009)

CTB-D2 D2 fibronectin-binding domain of Staphylococcus aureus fused with the cholera toxin B subunit

0.7% TSP (Dreesen et al., 2010)

10NF3, 14FN3

Domains 10 and 14 of human fibronectin, potential antibody mimics

14FN3: 3% TSP 10FN3:

detectable (Rasala et al., 2010)

M-SAA-Interferon β1

Multiple sclerosis treatment fused to M-SAA

Detectable (Rasala et al., 2010)

Proinsulin Blood sugar level-regulating hormone, type I diabetes treatment

Detectable (Rasala et al., 2010)

VEGF Human vascular endothelial growth factor isoform 121

2% TSP (Rasala et al., 2010)

HMGB1 High mobility group protein B1 2.5% TSP (Rasala et al., 2010)

MSP1 Vaccine against Malaria Detectable (Deauville et al., 2010)

Pfs25, Pfs28 Elicits Antibody that inhibit Malarial infection

Detectable (Gregory et al., 2012)


II. 4. 2. Strategies for foreign gene expression in C. reinhardtii

Three basic strategies are commonly used for the expression of foreign

protein in C. reinhardtii; i) Chloroplast transformation ii) Mitochondrial

transformation and iii) Stable nuclear transformation

II. 4. 2a. Chloroplast Transformation

Stable chloroplast transformation was first accomplished in 1988 when

Boynton and co-workers restored the photosynthetic capacity of a C. reinhardtii

mutant by cell bombardment with high-velocity micro-projectiles coated with the

wild-type gene. Once inside the organelle, the DNA integrates into the genome by

homologous recombination (Boynton et al., 1988). While the Chlamydomonas

chloroplast contains around 80 copies of its genome, homoplasmic transformants, in

which all genome copies contain the change, can be obtained after several rounds of

culturing on selective medium (Rochaix, 1997). Several approaches have been used

to select cells following chloroplast transformation and a detailed list of selectable

markers is presented in section. Early selection methods were based on cloned

chloroplast genes used to rescue photosynthesis mutants (Boynton et al., 1988).

Chloroplast genes that confer resistance to antibiotics or herbicides have also been

widely used (Newman et al., 1992). However, a major breakthrough in chloroplast

transformation came with the development of dominant selectable markers based on

bacterial genes for antibiotic resistance (Goldschmidt- Clermont, 1991).

Foreign protein expression in the chloroplast of C. reinhardtii for the first

time involved the bacterial neomycin phosphotransferase (Blowers et al., 1989) and

β-glucuronidase genes (Blowers et al., 1990), both driven by C. reinhardtii

chloroplast promoters. To date, only a handful of therapeutic proteins have been

expressed in algal chloroplasts. Production of recombinant proteins in chloroplasts

also possesses several unique attributes. At present transgenic proteins can

accumulate to much higher levels in the chloroplast than when expressed from the

nuclear genome, mainly because plastids lack gene silencing mechanisms and other

mechanisms that reduce recombinant protein production from nuclear encoded genes

42 Chapter II

(Bock, 2007). Chloroplasts can be transformed with multiple genes in a single event,

due to the availability of multiple insertion sites, as well as an ability to process

polycistronic transcripts, allowing an entire gene cassette to be regulated by a single

promoter (Rymarquis et al., 2006; Bock, 2007). Additionally, proteins produced

within the chloroplast are not glycosylated (Franklin and Mayfield, 2005), which can

prove useful in many applications such as producing antibodies that are similar to

native antibodies in their ability to recognize their antigen, but whose lack of

glycosylation prevents them from recruiting killer cells (Tran et al., 2009).

II. 4. 2b. Mitochondrial Transformation

The mitochondrial genome of C. reinhardtii has also been transformed using

the biolistic method. Function was restored to a respiratory-deficient C. reinhardtii

mutant (dum-1) with a 1.5 kb deletion in the apocytochrome b gene (cob) by

bombardment with partially purified total mitochondrial DNA (Randolph-Anderson

et al., 1993) and selection for growth in the dark on acetate-containing media. Since

the mitochondrial genome of C. reinhardtii is small and contains very few genes, it is

unlikely to be developed further as a system for genetic engineering.

II. 4. 2c. Nuclear Transformation

The nuclear genome of C. reinhardtii was first transformed using the biolistic

method over 23 years ago, making it the first photosynthetic organism in which both

the nuclear and chloroplast genomes were transformed (Debuchy et al., 1989;

Boynton et al., 1988). However, using this method, transformants were recovered at

consistently low levels and the foreign DNA was usually introduced into the genome

in multiple copies (Kindle et al., 1989). Today, glass beads have superseded

biolistics as the most commonly used method for the nuclear transformation C.

reinhardtii. This method is simple and yields high numbers of transformants, but it

requires cells lacking a cell wall. The nuclear genome of wild-type C. reinhardtii

cells has also been transformed using silicon carbide (SiC) whiskers (Dunahay,

1993) and electroporation (Brown et al., 1991).


Chloroplasts are generally preferred for foreign protein expression in

microalgae due to high expression levels. But nuclear expression is necessary for the

expression of complex proteins which require post-transcriptional and post-

translational processing and targeting despite low yields. Transgene expression from

the nuclear genome offers several advantages over chloroplast expression, such as

glycosylation and other post-translational modifications and heterologous protein-

targeting to sub-cellular locations or for secretion (Leon-Banares, 2004).

Nuclear expression of foreign proteins remains very low, for reasons that are

as yet not well understood. Positional effects, RNA silencing, a prohibitively

compact chromatin structure and non-conventional epigenetic effects have been

proposed as possible causes. Several advancements have been developed for

improved nuclear transgene expression. For nuclear-expressed proteins, degradation

can be minimized by targeting protein synthesis to the ER rather than to the cytosol

(Conrad and Fiedler, 1998), a strategy that lead to a 104-fold increase in recombinant

growth factor expression in tobacco (Wirth et al., 2004). A recent study describes the

selection of highly expressed nuclear transgenes following UV-induced mutations of

transformed strains. Using this protocol, yields of foreign proteins accounting for

0.2% total soluble protein (TSP) were achieved, which is relatively high for nuclear

expression (Neupert et al., 2009). A possible strategy to minimize proteolytic

degradation of proteins requiring modification would therefore be to target the

nuclear-expressed proteins to the chloroplast for storage (Potvin and Zhang, 2010).

Inserting introns from native genes in heterologous sequences under the control of

that gene's promoter has been shown to increase protein yields. Expression of

recombinant genes in the nuclear genome of C. reinhardtii also improved following

the insertion of the first RBCS2 intron, which has been shown to contain an enhancer

element (Lumbreras et al., 1998; Berthold et al., 2002).

Agrobacterium mediated genetic transformation of C. reinhardtii was

reported in 2004 by Kumar and co-workers (Kumar et al., 2004). The

Agrobacterium-mediated transformation method offers several advantages over

44 Chapter II

direct gene transfer methods including its feasibility to transfer large DNA

fragments, low copy number of transgene integration with little rearrangement,

preferential integration into transcriptionally active regions and its simplicity (Cha et

al., 2011). Recent advances made in the Agrobacterium mediated genetic

transformation technology offer great opportunities for higher and more consistent

transgene expression in C. reinhardtii.

II. 4. 3. Molecular Factors Influencing Transgene Expression in C. reinhardtii

Choice of suitable selectable marker genes, reporter genes and efficient

promoters are key factors governing transformation. These molecular tools can

greatly enhance, reduce or even silence transgene expression. The studies conducted

so far in this regard facilitated the improvement of Chlamydomonas transformation

studies in recent years.

II. 4. 3a. Selectable Marker Genes

An important aspect of transformation is the choice of an appropriate

selective agent and its corresponding resistance gene. Selectable marker gene may

encode an enzyme capable of either detoxifying a phytotoxic compound (negative

selection) or metabolising a substrate (e.g. a carbon source) that wild-type cells

cannot utilize (positive selection). In Chlamydomonas transformation studies a

number of homologous and heterologous selectable marker genes have been used for

both nuclear (Table II-5) and chloroplast (Table II-6) transformation. The

heterologous genes introduced into the nuclear genome of C. reinhardtii are often

poorly expressed. Possible reasons for this failure include: epigenetic suppression of

gene expression, inefficient transcription due to lack of the appropriate promoter

and/or enhancer elements, the lack of introns required for efficient RNA processing,

and poor translation of foreign mRNA due to biased codon usage (Kindle and

Sodeinde, 1994; Stevens et al., 1996; Cerutti et al., 1997a). The nptII gene was only

weakly expressed and direct selection for kanamycin resistance proved unsuccessful

(Hall et al., 1993).


Table II-5: Selectable marker genes used in C. reinhardtii nuclear transformation

Gene Gene product Selection method Reference Heterologous genes

AadA

Aminoglycoside 3’ adenyl transferase from E. coli

Spectinomycin, streptomycin

(Cerutti et al., 1997b) (Meslet-Cladiere and Vallon, 2011)

Als

Acetolactate synthase gene mutant

Resistace to herbicide sulphometuron methyl

(Kovar, 2002)

aphA

Aminoglycoside 3- phosphotransferase from Streptomyces rimosus

Kanamycin, Paromomycin, G418

(Hasnain et al., 1985)

aph7

Aminoglycoside 3- phosphotransferase from Streptomyces hygroscopicus

Hygromycin B

(Berthold et al., 2002)

aphVIII

Aminoglycoside 3- phosphotransferase from Streptomyces rimosus

Paromomycin

(Sizova et al., 2001; Hallmann and Wodniok, 2006)

ble

Bleomycin-binding protein of Streptoalloteichus hindustanus

Phleomycin, bleomycin, zeocin

(Stevens et al., 1996)

hpt

Hygromycin phosphotransferase of E. coli

Hygromycin

(Butanaev, 1994)

nptII

Neomycin phosphotransferase II of E.coli

Kanamycin, Geneticin

(Bingham et al., 1989; el- Sheekh, 2000)

Oee-1

Oxigen evolving enhancer protein

Photosynthetically incompetent to grow in the absence of Oee-1

(Mayfield and Kindle, 1990)

Homologous genes AC29

AC29 gene product (albino-3 homolog)

Complements yellow ac29 mutant

(Ferris, 1995; Ferris et al., 2002; Bellafiore et al., 2002)

Als

Acetolactate synthase

Resistance to sulfonylurea herbicides

(Funke et al., 1999; Kovar et al., 2002)

Arg7

Argininosuccinate lyase

Corrects arginine auxotrophy of arg7 mutant

(Debuchy et al., 1989; Kovar, 2002)

Cry1

Mutant cytosolic ribosomal protein S14

Cryptopleurine & emetine resistance

(Nelson et al., 1994)

Nic7 Guinolinate synthetane A

Complements a nicotinamide-requiring mutant nic7

(Ferris, 1995; Ferris et al., 2002)

Nit1 Nitrate reductase Complements nit1 mutant (Kindle et al., 1989)

Nit2 Nitrate regulatory gene Nitrate regulation (Ferris et al., 1996) Ppx1

Protoporphyrinogen oxidase Resistance to N-phenyl heterocyclic herbicides

(Randolph-Anderson et al., 1998)

Thi10

Hydroxyethyl thiazole kinaze Complements thiamine requiring mutant thi10

(Ferris, 1995; Ferris et al., 2002)

46 Chapter II

Table II-6: Selectable marker genes used in C. reinhardtii chloroplast transformation

Gene Gene product Selection method Reference Heterologous genes aadA


Spectinomycin, streptomycin

(Goldschmidt-Clermont, 1991)

aphA6

Aminoglycoside 3 phosphotransferase from Streptomyces rimosus

Kanamycin, Amikacin

(Bateman and Purton, 2000)

Cat

Chloramphenicol acetyltransferace

Resistace to chloramphenicol

(Brown et al., (1991)

nptII

Neomycin phoshotransferace gene from E. coli

Resistance to G418 (Butanaev, 1994)

nitl Nitrate reductase Nitrate reduction (Kindle et al., 1989) Homologous genes atpA,B,E

α-, β-, & ε-subunits of the CF1 ATP synthase complex

Restores Photosynthesis

(Lemaire and Wollman, 1989; Kindle et al. 1991; Leu et al., 1992; Boynton et al., 1988)

chlL/chlN

Protochlorophyllide reductase Yellow in dark

(Choquet et al. 1992; Suzuki and Bauer, 1992)

petA & D

Subunits I and IV of cytochrome b6/f complex

Restores photosynthesis

(Buschlen et al., 1991; Baymann et al., 1999)

psaA Photosystem I reaction centre protein

Restore photosynthesis

(Choquet et al., 1988)

psaB Photosystem I reaction centre protein


(Bingham et al., 1991)

psaC Iron sulphur protein of photosystem I

Restores Photosynthesis

(Takahashi et al., 1991)

psbA

Photosystem II reaction centre protein D1

Restores photosynthesis & confers DCMU resistance on wild type

(Przibilla et al., 1991; Hatano- Iwasaki et al., 2000)

psbC P6 photosystem II core protein


(Rochaix et al., 1989)

psbD D2 protein of photosystem II Restores photosynthesis

(Erickson et al., 1986)

rbcL Rubisco large subunit Restores photosynthesis

(Newman et al., 1991)

rps4 &rps12

Ribosomal protein S4 & S12

Streptomycin dependence to independence

(Liu et al. 1989; Randolph- Anderson et al. 1995)

tscA Photosystem 1 compex Restores photosynthesis

(Goldschmidt-Clermont, 1991)

16S rDNA

16S rRNA

Streptomycin, Spectinomycin, Kanamycin resistance

(Newman et al., 1990)

23S rDNA 23S rRNA Erythromycin (Newman et al., 1992)


The most commonly used selectable markers for Chlamydomonas nuclear

transformation are Chlamydomonas genes that complement mutations in the

corresponding structural genes. The most widely used markers have been Nit1,

which encodes nitrate reductase (Fernandez et al., 1989; Kindle et al., 1989), and

Arg7, which encodes argininosuccinate lyase (Debuchy et al., 1989). A mutated

version (Cry1) of the RPS14 gene encoding ribosomal protein S14, was the first

dominant selectable marker allowing direct transformation of wild-type

Chlamydomonas and confers resistance to the translation inhibitors emetine and

cryptopleurine (Nelson et al., 1994). Recently, transformation of C. reinhardtii

with a mutated acetolactate synthase gene (ALS) under the control of the native

RbcS2 promoter allowed recovery of colonies resistant to the herbicide

sulfometuron methyl (Kovar et al., 2002). Drug resistance markers that have also

been used in Chlamydomonas including bacterial genes that confer resistance to

bleomycin (ble gene encodes a bleomycin-binding protein) (Stevens et al., 1996)

and spectinomycin/streptomycin (aadA gene, encodes aminoglycoside adenine

transferase; Cerutti et al., 1997b). The bacterial aadA gene is also the most

commonly used dominant marker for C. reinhardtii chloroplast transformation

(Goldschmidt-Clermont, 1991).When fused to the 5` and 3` untranslated regions

of endogenous chloroplast genes it was shown to confer resistance to

spectinomycin and streptomycin in both Chlamydomonas (Goldschmidt-

Clermont, 1991) and higher plants (Svab and Maliga, 1993). Two strategies for

removal of the aadA cassette from the chloroplast genome have also been

developed (Fischer et al., 1996). The first relies on homologous recombination

between direct repeats flanking the aadA cassette to produce direct excision of

the cassette when selection is removed. A second is to co-transform with a

plasmid containing an essential chloroplast gene disrupted by the aadA

cassetteand with a plasmid containing a modified non-essential chloroplast gene.

Under selective conditions the second mutation is transferred to all chloroplast

copies while the aadA insertion remains heteroplasmic and is subsequently lost

when selection is removed. Another selectable marker gene is the bacterial

48 Chapter II

aminoglycoside 3-phosphotransferase gene (aphA6), which allows for the direct

selection of chloroplast transformants on selective media containing kanamycin

or amikacin and can also be used to modify or inactivate specific chloroplast

genes by insertional inactivation (Bateman and Purton, 2000).

II. 4. 3b. Promoters

One important aspect in the development of a transformation system has

been the choice of promoter to drive expression of the transgene. One of the most

widely used promoters in plant molecular biology is the cauliflower mosaic virus

35S (CaMV 35S) promoter. Although the CaMV 35S promoter drives strong and

constitutive expression in most dicotyledonous and some monocotyledonous

plants (Benfey et al., 1990), it has not shown to be a useful promoter in most

algal species. Algal transformation has been most successful using promoters

derived from highly expressed algal genes (Table II-7). A widely used promoter

for Chlamydomonas transformation is derived from the 5’ untranslated region of

the C. reinhardtii ribulose bisphosphate carboxylase/oxygenase small subunit

(RbcS2) (Stevens et al., 1996). It was also shown that transformation frequency

was significantly increased when Chlamydomonas introns (particularly the first

intron of RbcS2) were introduced into the coding region of the ble selectable

marker gene (Lumbreras et al., 1998). This intron appears to contain a

transcriptional enhancer element as it can act in an orientation-independent

manner and is effective when placed either upstream or downstream of the

promoter. Synthetic promoters have also been developed by fusing the promoter

from the Chlamydomonas Hsp70A (heat shock protein 70A) gene to other

Chlamydomonas promoters. The Hsp70A promoter serves as a transcriptional

enhancer of promoters RbcS2, β2-tubulin and Hsp70B leading to high-level

expression under inducing conditions (Schroda et al., 2000).


Table II-7: Promoters shown to drive transgene expression in C. reinhardtii

Promoter Derivation Regulation Reference

Nuclear

Amt

Chlorella virus adenine methyltransferase

Constitutive

(Kang et al., 2000)

Ca1/ Ca2 Carbonic anhydrase CO2 and light (Villand et al., 1997)

CabII-1

Chlorophyll a/b-binding protein of photosystem I

Light

(Blankenship and Kindle 1992)

CaMV 35S

Cauliflower mosaic virus 35S promoter

Constitutive

(Kumar et al., 2004; Ruecker et al., 2008)

Hsp70A Heat shock protein 70A Heat shock, light

(Schroda et al., 2000)

Hsp70B Heat shock protein 70B Heat shock, light

(Schroda et al., 2000)

Nia1 (Nit1)

NAD(P)H nitrate reductase

Light, nitrate

(Ohresser et al., 1997; Loppes et al., 1999)

Nos

Agrobacterium tumefaciens nopaline synthase

Constitutive

(Hall et al., 1993)

PsaD Photosystem II subunit D Light (Fischer and Rochaix, 2001)

RbcS2

Small subunit of ribulose-1,5 bisphosphate carboxylase/

Oxygenase

Light

(Stevens et al., 1996)

TubB2 β2-tubulin Deflagellation (Davies et al., 1992)

Hsp70A-RbcS2

Heat shock protein 70A- RbcS2

Heat shock, light

(Wu et al., 2008)

SV40 Animal adenovirus SV40 Constitutive (Butanaev, 1994)

Chloroplast

atpA

α-subunit of the ATP

synthase CF1 complex

Light

(Blowers et al., 1990)

atpB

β-subunit of the ATP

synthase CF1 complex

Light

(Blowers et al., 1990)

chlL UV resistance Light (Zang et al., 2006)

petD

Subunit IV of

cytochrome b6/f complex

Light

(Sakamoto et al., 1993)

rbcL

Large subunit of ribulose

bisphosphate carboxylase

Light

(Klein et al., 1994)

rrn16 16S rRNA Light (Klein et al., 1992)

50 Chapter II

II. 4. 3c. Reporter Genes

Transformation systems are generally developed using an efficient

reporter gene, which encodes a protein that can be easily detected and quantified

in transgenic lines. The most commonly used reporter genes in higher plants are

uidA, which encodes β- glucuronidase (GUS) and the green fluorescent protein

gene (gfp) from the bioluminescent jellyfish Aequorea victoria (Chalfie et al.,

1994). There has been considerable difficulty to date in the expression of

heterologous reporter genes in the nuclear genome of Chlamydomonas. The uidA

gene has not been a successful reporter gene for nuclear transformation of C.

reinhardtii even when under the control of a native promoter (Blankenship and

Kindle, 1992).

The use of GFP as a reporter gene in both the nuclear and chloroplast

genome of Chlamydomonas has been demonstrated (Fuhrmann et al., 1999;

Franklin et al., 2002). Unfortunately, the unmodified GFP gene under the control

of heterologous promoters gave poor expression when used as a reporter for

nuclear transformation of Chlamydomonas (Fuhrmann et al., 1999). Reasons for

poor expression may include the predominance of A/T rich codons in the native

gene and the possible presence or absence of sequence motifs that regulate

transcription or the processing and targeting of the transcript. To overcome these

limitations a modified gfp gene has been synthesised utilising the codon

preference of C. reinhardtii nuclear genes (Fuhrmann et al., 1999). Additional

modifications known to modify the spectral properties of GFP-protein for

fluorescence imaging were also introduced. This modified gene was fused in-

frame to the ble gene and expressed under the control of the RbcS2 promoter.

GFP fluorescence was observed in the nucleus demonstrating nuclear

accumulation of the GFP-BLE fusion protein (Fuhrmann et al., 1999). GFP-

fusions have proven to be a useful tool for the study in vivo of dynamic processes

such as flagellar and centriole assembly and cell cycle events in Chlamydomonas

(Ruiz-Binder et al., 2002).


A modified gfp gene for Chlamydomonas chloroplast transformation has

also been synthesized by optimizing its codon usage to reflect that of the major

chloroplast encoded proteins (Franklin et al., 2002). Chloroplasts transformed

with this modified gene under the control of the C. reinhardtii rbcL 5’- and 3’-

UTRs were shown to accumulate ~80-fold more GFP than those transformed

with the native gene. The GFP accumulated to ~0.5% of the total soluble protein

(TSP), ~50-fold higher than reports of uidA (GUS) expression under the control

of the same promoter (Ishikura et al., 1999). In contrast to the high-levels of GFP

visible in the nucleus GFP fluorescence in the chloroplast was not easily

visualized using fluorescence microscopy (Franklin et al., 2002). This is most

likely due to the chlorophyll and other pigments within the chloroplast absorbing

much of the incident light targeted to GFP, or some of the light being emitted by

the GFP being reabsorbed by the chloroplast pigments. GFP has been visualized

in the chloroplasts of higher plants (Reed et al., 2001), but only when the levels

of GFP accumulated to over 5% of the TSP, approximately 10-fold higher than

levels presently obtained in C. reinhardtii. The most encouraging development in

chloroplast transformation is the recent report of successful GFP visualization in

the Chlamydomonas chloroplast using a gfp gene that had been modified for

tobacco chloroplast expression (Komine et al., 2002).

The most commonly used reporter genes for Chlamydomonas

transformation are homologous genes (Table. II-8). Two Chlamydomonas genes

confer easily discernible phenotypes. The ARS, gene encodes the periplasmic

enzyme arylsulfatase, which is normally expressed only under conditions of

sulphur starvation. Arylsulfatase activity can be detected in colonies by spraying

plates with the chromogenic substrate 5- bromo-4-chloro-3-indolyl sulphate

(XSO4) or assayed in solution with p-nitrophenyl sulphate or α-naphthylsulphate

(Davies et al., 1992). The second commonly used reporter gene PC1, encodes

NADPH: protochlorophyllide oxidoreductase (POR) and is responsible for light-

dependent protochlorophyll reduction (Li and Timko, 1996). Wild-type strains of

Chlamydomonas make chlorophyll and are green when grown in both the light

52 Chapter II

and the dark. Strains containing mutations in both PC1 and in genes of the light-

independent protochlorophyllide-reduction pathways are unable to produce

chlorophyll under any light conditions. After transformation with pc1 they form

green colonies in the light only.

Table II-8: Commonly used reporter genes for transformation studies in C. reinhardtii

Gene Gene Product Assay Method Reference

Nuclear

Ars

Arylsulfatase

Chromogenic detection using X-SO4 as a substrate

(Davies et al., 1992)

GFP

Green fluorescent protein of Aequora victoria

Luminescence or antibody based

(Fuhrmann et al., 1999)

PC1

NADPH: protochlorophyllide

Oxidoreductase

Assay protochlophyllide

reductase mRNA or enzyme activity

(Li and Timko., 1996)

Cgluc luciferase of Gaussia princeps Luminescence (Ruecker et al., 2008)

Rluc

luciferase of Renilla reniformis

Luminescence (Fuhrmann et al., 2004)

Chloroplast

aadA


AAD assay

(Zerges et al., 1997)

GFP

Green fluorescent protein of Aequora victoria

Fluorescence or antibody based

(Franklin et al., 2002; Komine et al., 2002)

LUC

Luciferase from Renilla reniformis

Luminescence or antibody based

(Minko et al., 1999)

uidA

β-glucuronidase of E. coli

Histochemical or flurometric

(Sakamoto et al., 1993; Ishikura et al., 1999; el-Sheekh 2000)

II. 4. 4. Methods used for the transformation of C. reinhardtii

Appropriate transformation method used for the delivery of foreign DNA

to target microalgal genome greatly influences transformation efficiency and

expression. Research works over the years have developed in a number of


methods for genetic manipulation of C. reinhardtii making it a top candidate for

biotechnological applications (Harris, 1989; Fuhrmann, 2002). Although some of

these methods may not have significantly changed since their initial development,

they are still being applied and studied. Recently a number of studies have

presented methods that seem to offer advantages over earlier techniques.

II. 4. 4a. Cell wall-deficient strains

The use of cell wall-deficient strains, or the removal of the cell walls from

wild-type strains, greatly increases the number of transformants recovered

following transformation. Protocols for cell wall removal have been developed

which facilitate the study of microalgae. These protocols involve the mating of

mating type plus (mt+) and mating type minus (mt−) gametes of C. reinhardtii.

The specific cell–cell recognition resulting from flagellar interaction leads to the

release of enzymes, autolysin or lysin, that cause cell wall degradation. These

enzymes can be purified and used as a pre-treatment to transformation. A detailed

protocol for production and purification of these enzymes is given by Buchanan

and Snell (1988) and a detailed study of the mating process was more recently

reported by Hoffmann and Beck (2005).

II. 4. 4b. Particle bombardment

Bombardment of target cells with DNA-coated metallic particles is a

widespread, simple, effective and highly reproducible transformation method.

This method has been successfully employed for the transformation of most

standard cellular expression systems, and it is therefore not surprising that it is

also useful for the study of microalgae. The main drawback of the particle

bombardment method is the cost of the required specialized equipment. Although

the number of transformants recovered following particle bombardment can be

low, it remains the most effective method for the transformation of chloroplasts,

as it allows for the delivery of multiple copies of recombinant DNA through both

the cellular and chloroplast membranes, increasing the chance for a successful

integration event to occur (Boynton and Gillham, 1993). This method has been

54 Chapter II

shown to be effective for the stable nuclear (Mayfield and Kindle, 1990) and

chloroplast (Boynton et al., 1988; El Sheekh, 2000; Ramesh et al., 2011)

transformation of C. reinhardtii, the transformation of Volvox carteri

(Schiedlmeier et al., 1994), Chlorella sorokiana (Dawson et al., 1997), C.

ellipsoidea (Chen et al., 1998) and C. kessleri (El-Sheekh, 1999) species,

transient transformation of H. pluvialis (Teng et al., 2002) and the stable nuclear

transformation of the diatom Phaeodactylum tricornutum (Apt et al., 1996).

Recent work has shown that the particle bombardment method is also effective

for the transformation of more complex algal species, such as the multicellular

Gonium pectorale (Lerche and Hallmann, 2009).

II. 4. 4c. Glass beads method

A simple and effective transformation method consists of agitating cell

wall-deficient microalgal cells with recombinant DNA, polyethylene glycol

(PEG) and glass beads, which greatly increases transformation efficiency.

Despite the drop in cell viability to 25% following agitation with the beads, a

nuclear transformation efficiency of about 103 transformants/µg DNA was

achieved using this method (Kindle, 1990) and an efficiency of 50

transformants/µg DNA was achieved for the transformation of C. reinhardtii

chloroplasts (Kindle, 1990). Compared to the particle bombardment method, the

glass beads method is simpler, more efficient for nuclear transformations, and

much less expensive as it does not require specialized equipment. A recent study

showed that the glass beads method is also more efficient than particle

bombardment for the transformation of Dunaliella salina (Feng et al., 2009).

II. 4. 4d. Silicon carbide whisker method

A similar protocol, using silicon carbon whiskers instead of glass beads to

pierce cells, has also been used successfully (Dunahay, 1993; Wang et al., 1995).

Cells are transformed by mixing with the SiC whiskers and DNA, and vortexing

briefly. Although the exact mechanism for whisker-mediated transformation is

unknown, it is known that fractured SiC crystals readily form sharp cutting edges.


The surface of SiC whiskers is negatively charged which probably results in there

being little affinity between whiskers and DNA in a neutral pH medium. It has

been suggested therefore that the whiskers do not carry the DNA into the treated

cells but function as needles that facilitate DNA delivery by cell perforation and

abrasion during mixing (Wang et al., 1995). The cell viability following agitation

is much improved, but due to low transformation efficiencies, high cost of

materials, and health concerns associated with the handling of the whiskers, the

glass beads are generally preferred. However, SiC whiskers have since been

reported to be extremely hazardous to humans and therefore are rarely used.

II. 4. 4e. Electroporation

Electroporation or electropermeabilization is a transformation technique

that use induction of macromolecular uptake by exposing cell walls to high

intensity electrical field pulses,. The effectiveness of microalgal electroporation,

was first reported by Brown et al., (1991). Electroporation specifically disrupts

lipid bilayers, leading to efficient molecular transport across the plasma

membrane (Azencott et al., 2007). Efficient electroporation mediated

transformation was achieved in both wild-type and cell wall-deficient strains

(Brown et al., 1991). The transformation efficiency of electroporation is two

orders of magnitude higher than the glass beads method, and only requires

relatively simple equipment (Shimogawara et al., 1998). Important parameters

affecting the effectiveness of electroporation include field strength, pulse length,

medium composition, temperature and membrane characteristics (Brown et al.,

1991) as well as the concentration of DNA (Wang et al., 2007a). Electroporation

was successfully used for the transformation microalga D. salina (Geng et al.,

2004; Sun et al., 2005; Wang et al., 2007b; Sun et al., 2008; Feng et al., 2009),

D. viridis (Sun et al., 2006) and D. tertiolecta (Walker et al., 2005b), C.

reinhardtii (Tang et al., 1995; Shimogawara et al., 1998; Kovar et al., 2002;

Ladygin, 2003, 2004), Chlorella sp. (Chow and Tung, 1999; Wang et al., 2007a),

and Nannochloropsis oculata (Chen et al., 2008; Li and Tsai, 2009).

56 Chapter II

II. 4. 4f. Agrobacterium tumefaciens-mediated transformation

Agrobacterium tumefaciens is widely used for genetic transformation of

plants because of its natural ability to transfer foreign DNA into the host plant

genome. It is also a significant plant pathogen that causes crown gall disease on

several agronomically important species, including grape vines, stone fruit, and

nut trees (De Cleene and De Ley, 1976). Transformation results from the

stimulation of cell division by products encoded by T-DNA transferred from

Agrobacterium to the target cell. The T-DNA and virulence (vir) regions are

located on the tumour inducing plasmid (pTi). The vir system processes and

transfers any DNA between the short flanking repeats that delimit the T-DNA

(R&L T-DNA borders), making Agrobacterium an efficient DNA delivery

system (Akhond and Machray, 2009).

The process of T-DNA transfer is initiated by the induction of bacterial

virulence (vir) genes by the phenolic compounds produced and released by the

wounded plant cells through the VirA–VirG two-component signal transduction

system (Zupan et al., 2000). It has been shown that majority of the

dicotyledonous plants produce such phenolic compounds and they have been

routinely transformed by Agrobacterium (Gelvin, 2000). Monocotyledons, which

generally do not produce vir-inducing compounds, could be transformed by the

exogenous addition of such molecules like acetosyringone (AS) (Hiei et al.,

1997). Agrobacterium gene transfer has also been extended to fungi (De Groot et

al., 1998) and human (HeLa) cells (Kunik et al., 2001).

The prospects of A. tumefaciens mediated transformation were first

reported in microalgae by Kumar and co-workers (Kumar et al., 2004). The

microalga C. reinhardtii was successfully transformed with uidA (β-

glucuronidase), gfp (Green Fluorescent Protein) and hpt (hygromycin

phosphotransferase) genes in presence of acetosyringone (AS). Later Kumar and

Rajam reported successful transformation of C. reinhardtii even in the absence of

AS (Kumar and Rajam, 2007), with about fifty-fold increase in transformants


compared to the commonly used glass beads method for genetic transformation in

C. reinhardtii.

The major difficulty associated with the nuclear transformation of C.

reinhardtii was gene silencing as in many other organisms (Manuell and

Mayfield, 2006). Agrobacterium mediated transformation has been reported to

overcome the major limitations of other transformation procedures, providing

stable integration at lower copy number, potentially leading to fewer problems

with transgene co-suppresion, instability (Hansen et al., 1997). Integration of

transgenes into the nuclear genome of C. reinhardtii and their expression without

any silencing was obtained in the studies conducted by Kumar and co-workers

(2004, 2007). This can be attributed to the possible advantages of Agrobacterium

T-DNA to be targeted to and integrated at potentially transcribable regions of the

genome (Hiei et al., 1994). Similar promising results were observed in the

Agrobacterium mediated genetic transformation of other microalgal species,

Haematococcus pluvialis (Kathiresan et al., 2009), Dunaliella bardawil (Anila et

al., 2011), Nannochloropsis sp (Cha et al., 2011), Schizochytrium (Cheng et al.,

2012) and Chlorella vulgaris (Cha et al., 2012).

Though different methods are available for the genetic transformation of

C. reinhardtii, like particle bombardment, glass beads, silicon carbide etc,

transformation frequency and stability of gene in subsequent generations have

been a limiting factor. But in case of Agrobacterium mediated transformation,

cell lines showed high stability in gene integration for up to 18 months in C.

reinhardtii (Kumar et al., 2004).

The Agrobacterium mediated genetic transformation technique is a

simple, stable and efficient method for transgene experiment studies.

Undoubtedly, the recent development of this transformation procedure is

economically valuable for microalgal species and the results obtained in recent

years evidence a promising future. Taking these facts into consideration the

present work was taken up to develop C. reinhardtii as an expression system for

58 Chapter II

the production of recombinant human serum albumin (rHSA) through

Agrobacterium mediated genetic transformation.

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