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    Recombinant Human Growth Hormone

    BioE 110 -Project 1

    Due: October 5, 2010

    Steven Grillo, Rachel Nordberg, Ilan Beitscher, Peter Sords

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    1. Introduction

    With the advent of recombinant DNA technology, the controlled

    microbial production of an enormous variety of useful polypeptides has

    become possible. One such product of the technology that is currently onthe market as well as in use is the recombinant human growth hormone.

    Somatropin, or more commonly known as recombinant human growth

    hormone (rHGH), is a synthetic growth hormone. Somatropin is a protein

    hormone that stimulates growth in the human body along with cell

    reproduction and regeneration. Many years ago, inserting HGH into your

    body was an ethical and health issue, which discouraged the use by many

    people. The health issue was the more significant concern in that the

    hormone was attained through the pituitary gland of a cadaver, and hence,

    caused infection and disease. Biotechnology became the answer to this

    problem. Recombinant DNA technology was used to create the recombinant

    human growth hormone. It took until 1985 for Somatropin to replace

    pituitary derived HGH. Today, all human growth hormone that is being used

    is created via recombinant DNA technology.

    The first use of recombinant HGH on humans occurred in 1981 by

    Genentech. However, there is an extremely interesting case that developed

    in the years of Genetechs initial creation. Allegedly, Genetech stole

    research materials that were in the laboratories at the University of

    California-San Francisco. They were blamed of sneaking into their

    laboratories at midnight on New Years Eve and simply stealing materials

    from the Universitys scientists. There was an extremely long trial that took

    place and ended in Genetech having to pay the University $150 million and

    donate $50 million towards a new research building. This case led to larger

    arguments about genes and gene sequences and their patentability

    (Rimmer).

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    Somatropin, identical to the growth hormones in the human body, is a

    22,000 Dalton and 191 amino acid sequence peptide produced during

    fermentation in E.coli. Initially a 192 amino acid sequence is formed, but

    the methionine is removed to create an exact match to the pituitary derived

    human growth hormone. The growth hormone works by a process of

    secretion by the somatotrophs of the anterior pituitary gland. It then

    enables growth within the body due to interaction with various tissues.

    Synthetic HGH is used to treat individuals who suffer from growth

    hormone deficiency. HGH has many functions including tissue repair, muscle

    growth, bone strength, brain function, and metabolism. An individual with a

    deficiency of growth hormone can be abnormally short in stature and

    maintain a slow growth rate and is therefore diagnosed with pituitary

    dwarfism. There are many cases in society in which a doctor would

    recommend that a child

    take human growth

    hormone so that the

    individual can have the

    ability to be at a more

    typically normal height.

    HGH is produced by

    means of recombinant

    DNA technology and is

    used for treatment of

    pituitary dwarfism in the

    field of pediatrics.

    However, synthetic human growth hormone is misused greatly by athletes

    and others around the globe. HGH is not supposed to act as enhancement

    therapy or as a booster for additional growth for an individual who is

    already considered or projected to be normal height. Unfortunately, it is not

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    the case that this is only used for treatment. This is reflected by the

    numerous doping or steroid problems within professional sports. Athletes

    use HGH to enhance their individual performance in their respective sports

    to gain a competitive advantage.

    HGH is currently on the market and is a drug that should be prescribed

    by a medical doctor. As of 2005, recombinant growth hormones available

    in the United States included Nutropin (Genentech), Humatrope (Lilly),

    Genotropin (Pfizer), Norditropin (Novo), and Saizen (Merck Serono). This is

    a drug that has an affect on humans each and everyday. It can be a minute

    reason such as a favorite baseball player caught doing steroids or a more

    serious scenario if you are a person that may be suffering from human

    growth hormone deficiency and need to consider taking HGH. Somatropin

    treats a variety of conditions of growth failure and growth retardation. A

    major group of people in which rHGH can help are those who suffer from

    growth hormone deficiency, Turners syndrome, and short stature according

    to skeletal age. Recombinant DNA technology opens the way to the large-

    scale commercial production of human growth hormone, and the

    recombinant HGH appears to have equivalent biological efficacies and

    pharmacokinetic properties. Recombinant technology has led to advances

    in protein production and classification, and therefore has increased the

    therapeutic applications of proteins and will hopefully continue to have an

    impact in the future.

    2. Biotechnology

    2.1 Characterization of Human Growth Hormone

    Human growth hormone (hGH) is secreted in the human pituitary, a pea-

    sized gland located at the base of the brain. The molecular weight of hGH is

    about 22,000 (22K form) and it consists of 191 amino acids (or residues)

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    with two loops formed by disulfide bridges (see

    Figure 6). The molecule is predominantly -

    helical in secondary structure.

    Involved in metabolism, growth, and lactation,

    GH can have both anabolic and catabolic

    effects: while it increases lean mass by

    increasing muscle and bones, it reduces fat.

    Some effects are mediated directly, others are

    mediated indirectly through the action of

    somatomedins hGH-dependent growth

    factorssuch as insulin growth factor 1 (IGF-1). Figure 1 illustrates hGH

    (top left) and IGF-1 (bottom) proteins,

    along with prolactin (top right), another

    pituitary hormone closely related to

    hGh. Because of its pervasive role in the

    growth of both soft and skeletal tissue,

    human growth hormone is of

    considerable medical importance, and

    its cloning will provide boundless

    benefits.

    2.2 The construction and expression

    of a cloning vehicle for human

    growth hormone

    Since hGH is a non-glycosylated protein, prokaryotic expression systems

    such as Escherichia coli have been preferred in the production of

    recombinant hGH. In 1979, scientists at Genetech produced human growth

    hormone by inserting DNA coding for human growth into a plasmid that was

    Figure 1

    Figure 2

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    implanted in E. colibacteria (Goeddel and Heyneker). This was first done the

    following way:

    I. Cloning the Hae III fragment of the mRNA transcript (Figure 2)Polyadenylated mRNA for human growth hormone was prepared from

    pituitary growth hormone tissue. A double strand (ds) cDNA was prepared

    from this RNA. The restriction pattern of hGH is such that Hae III restriction

    sites are present in th 3 noncoding region and in the sequence coding for

    amino acids 23 and 24 of hGH. Treatment of ds hGH cDNA with Hae III gives

    a DNA fragment of 551 base pairs (bp) coding for amino acids 24-191 of

    hGH. pBR322 was chosen as the cloning vehicle for the cDNA.

    Plasmid pBR322, shown in

    Figure 3, is a widely used

    plasmid and has been

    completely sequenced, with

    a known size of 4363bp.

    The most useful aspect of

    the DNA sequence is that it

    characterizes pBR322 in

    terms of its restriction

    sites, such that the exact

    length of every fragment

    can be calculated. There

    are 40 enzymes with

    unique cleavage sites on the pBR322 genome. pBR322 is a convenient

    cloning vehicle for hGH not only because it is a multicopy replicating

    plasmid, but also because it exhibits both ampicillin and teracycline

    resistance owing to its inclusion of the corresponding genes (ApR and

    Figure 3

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    TcR, respectively) and

    contains recognition sites for

    the restriction enzymes Pst I,

    EcoRI and Hind III.

    A GC tailing mehod was then

    employed to combine products

    od Pst I cleavage of pBR322

    and of Hae II digestion of the

    mRNA transcript, inserting the

    cDNA fragment into the Pst I

    site of pBR322 in such a

    manner as to restore the Hae

    III restriction sites on the

    cDNA while also restoring the

    Pst I restriction sites at each

    end of the insert. After annealing of the dC-tailed ds cDNA with the dG-tailed

    vector DNA, the mixture was first amplified using PCR, and then used to

    transform E. Coli x1776. The resulting plasmid, given the name pHGH31

    cloned in x1776 was analyzed using DNA sequence anylisis and was

    confirmed to contain the codons for amino acids 24-191 of hGH.

    II. Construction and Cloning of the Synthetic Gene Fragments (Figure 4)III. Construction of Plasmid for the Bacterial Expression of hGH (Firgure

    5)

    With the synthetic fragment in pHGH3 and the mRNA transcript in pHGH31,

    a replicatable plasmid containing both fragments was constructed using the

    expression plasmid pGH6, shown in Figure 5. The expression plasmid pGH6

    containing tandem lac UV5 promoters, was treated successively with Hind

    Figure 4

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    III, nuclease S1, and Eco RI and purified by gel electrophoresis. The

    resulting vector was ligated to the 591 bp hGH DNA.

    The ligation mixture was then to transform E. Coli x1776. Colonies were

    selected for growth on tetracyclin. It is noteworthy that insertion of the

    hybrid hGH gene into pGH6 destroys the promoter for the tetracyclin

    resistance gene, but that the tandem lac promoter permits read-through of

    the structural gene for tet resistance, retaining this selection characteristic.

    Transformants are then obtained and filter hybridization distiguishes colonies

    Figure 5

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    containing hGH sequences. The DNA sequences of one clone, pHGH107, was

    determined.

    2.3 Structure of hGH

    When engineering a novel protein

    for large-scale production in

    vitro, structure of the protein

    itself is of critical importance. The

    factor underlying the proteins

    conformation is its amino acid sequence. Figure 6 displays the amino acid

    sequence of hGH that any successful recombinant DNA should accurately

    translate. Any significant change in amino acid sequence could render the

    hGH protein less functional or even completely inactive. For example,

    changing a single glycine at position 120 to arginine will inactivate the hGH

    by not allowing it to bind to its receptor.

    As mentioned before, the experimentally

    determined protein structure of hGH

    predominantly consists of -helices. More

    specifically, hGH contains four helices 21-30

    residues long that are arranged in a left-

    handed bundle. The topology in unusual inthat the first two helices (beginning at the N-

    terminus) are parallel to each other and

    antiparellel to the last to (Wells and

    Abraham). To achieve this arrangement, long crossover connections link the

    Figure 6

    Figure 7

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    two sets of parallel helices, and a sort segment connects helix 2 to helix 3.

    Dilsulfides connect C53 in the first crossover connection to C164 in helix 4,

    and C181 in helix 4 to C189 near the C terminus.The core of hGH is almost

    exclusively made up of hydrophobic side chains. Figure 7 shows the hGHs

    tertiary structure found on Protein DataBank.

    2.4 Hormone ligand-receptor complex

    Growth hormone, shown in red in Figure 8,

    performs its multiple regulatory functions by

    binding to growth hormone receptors (shown in

    blue and green) on its target organs and cells.

    Growth hormone receptors are single-pass

    transmembrane receptors, with a three-domain

    architecture: an extracellular domain that binds

    to the activated ligand, a helical transmembrane

    segment, and a domain within the cytoplasm.

    Interestingly, growth hormone must bind to two

    receptor molecules simultaneously to mediate its funtion. This discovery thatactivation of the GH receptor requires dimerization can be used to produce

    receptor-specific analogues and to design receptor agonists and antagonists.

    (ex. Antagonist useful for treatment of acromegaly, a disease caused by the

    overproduction of hGH secreted from the pituitary.

    3. Bioprocessing

    3.1 Production of Plasmid

    The host cell used for the transformed production of hGH will be E. coli

    BL 21. The plasmid pBR322 will be used for the insertion of the hGH gene

    (figure 3). The hGH gene will be inserted with the use of the restriction

    enzymes EcoRI and HindIII. The restriction enzymes will digest the plasmid

    Figure 8

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    leaving sticky ends that will then adhere to the hGH gene. The insertion of

    the hGH gene would destroy the promoter for the gene that causes the E.

    coli to be resistant to tetracycline. Thus, using a tetracycline plate will allow

    for separation between the cells with the hGH gene and those without. The

    colonies that contain the desired hGH positive vector will be selected for

    fermentation.

    3.2 Fermentaion of E. Coli cells

    In order to create enough E. coli for cultivation of the hGH protein, a

    batch reactor growth scheme must be implemented. The fermentation in this

    process will take place at a pH of 7 and a temperature of 37C because that

    is the ideal condition for E. coli to grow. The pH will be maintained by the

    addition of ammonium hydroxide and phosphoric acid, as needed. The feed

    into the bioreactor will include glucose as a carbon source, anti-foaming

    agents, water, yeast as a nitrogen source, and trace amounts of B-D-

    thiogalactopyranoside (IPTG) to induce expression. Furthermore, a mixture

    of nutrients, trace metals, and inorganic salts will be added to the mixture in

    order to optimize E. coli growth. A mixture such as MaxyBroth manufactured

    by BioProgen Co. may be used (Shang). These reagents will all be added to

    a stirred tank reactor so that everything will be mixed together evenly.

    There will be high purity oxygen bubbled through the solution at

    concentrations higher than 30% in order to optimize the E. coli growth

    (Shang). The carbon source for growing the E. coli will be glucose, which will

    be added to the reactor at a rate slow enough to not exceed the oxygen

    supply and consequently, avoid the formation of acetic acid. The outflow of

    the reactor will be an E. coli rich solution containing rhGH ready to be

    processed.

    3.3 Separation of Inclusion Bodies

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    After fermentation, the cells must be separated from the batch

    fermentation medium. There will be inclusion bodies within the E. coli

    because the hGH hormone is very large. In order to isolate the inclusion

    bodies a three-step process will be used. First, the E. coli cells will be

    isolated from the rest of the mixture by centrifugation for 5 minutes at 5000

    rmp. This is a slow speed centrifugation so that the cells will not be

    damaged. The E. coli cells are large and hence, they will form a pellet at the

    end of the centrifugation. Once the pellet is formed the media will be

    decanted off. The cells were then re-suspended in a lysis buffer so that that

    there is a clean solution for the cells to release their inclusion bodies. The

    inclusion bodies will contain the recombinant hGH. The cells will then be

    shocked by rapidly cooling them to 0C. This is done so that the cells will

    lyse and inclusion bodies will be released into the solution. The lysis will be

    followed by a second centrifugation for 5 minutes at 10,000 rpm in order to

    isolate the inclusion bodies for the cell debris. This is a faster centrifugation

    because the inclusion bodies are smaller than the cells and will not break as

    easily as the cells. Again, the inclusion bodies and other large particles in

    solution will form a pellet at the bottom of the centrifugation tube and theliquid media will need to be decanted.

    3.4 Processing of rhGH protein

    The inclusion bodies containing rhGH will be suspended in a tris-HCl

    buffer solution. The inclusion bodies will go through chemical conversions in

    order to become the desired protein conformation of rhGH. In order to do

    this, a cleavage reaction will be induced using urea and guanidine

    hydrochloride because rhGH is a fusion protein. As a fusion protein, multiple

    genes were artificially combined to code for the protein. The two parts of the

    protein must be separated and thus will be cleaved apart during the

    reaction. Anion exchange chromatography will be used to separate the

    proteins knowing that rhGH is negatively charged. Once the proteins are

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    separated, sulfitolysis will be used to reform the disulfide bonds. This

    process will be done at a pH of 7 at room temperature because rhGH is

    highly sensitive to heat. Heat will cause the protein to denature again so the

    renaturing of the protein must not be in a heated system. Diafiltration will

    then be used to remove small ions and molecules left over from the chemical

    conversion steps. The diafiltration will get rid of some of the excess ions and

    molecules leaving the protein of interest and larger molecules. The rhGH will

    then be extracted from the diafiltration device and be ready to start

    chromatography.

    3.6 Chromatography Methods

    Once the protein is refolded, a series of chromatography methods will

    be used to separate the protein from the impurities. First, a size exclusion

    gel filtration chromatography will be used to remove small fragments and

    salts in the solution and to let the protein pass. The ions and small debris

    will be trapped in the matrix while larger molecules like the target protein

    rhGH will be able to cross the gel. Once the solution passes through the

    column, the lipids, glucose, and trace amounts of ions will still be in the

    solution with the rhGH. Hydrophobic interaction chromatography will help

    remove salts and cause hydrophobic non-polar fat molecules to aggregate.

    To completely

    separate hydrophobic

    and hydrophilic

    proteins, a reverse

    phase chromatography

    is performed. The

    hydrophilic outer

    portion of rhGH will

    not bind to theFigure 9

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    stationary hydrophobic phase and therefore remove other hydrophobic

    proteins from the mixture. In order to separate the protein from the rest of

    the solution ion exchange chromatography will be used. The rhGH protein is

    negatively charged. Therefore, an anion exchange using a positively

    charged column will capture the rhGH, but allow other impurities to flow

    through. The hydrophilic functional groups on the outside of rhGH will

    interact with the charged beads in the column. The nonpolar lipids and

    sugars will go straight through the column while the hGH protein stays in the

    column as the stationary phase. Ultrafiltration, the final filtration process,

    will suspend the large molecule of rhGH while allowing any remaining salt,

    water, and minerals left in solution to pass through the membrane. Figure 9

    shows what a protein gel might look like after various stages of

    chromatography with A being prior to chromatography and C being after

    many stages of chromatography. Once the rhGH is pure, it is ready to be

    recrystallized for storage.

    3.7 Recrystallization of rhGH protein

    After filtration is complete a recrystallization process must be carried

    out to make the biosynthetic drug a solid. Although at this time insulin is the

    only human therapeutic protein that is crystallized for administration, there

    are many advantages to the recrystallization of the protein such as extended

    shelf life and increased potency (Crisman). The recrystallization, like the

    renaturing steps earlier, will need to be preformed at room temperature so

    that the protein does not denature again. A nucleation agent will be added to

    the rhGH protein and the protein will become a solid, which will be able to

    last on the shelf for months until it is administered for clinical use.

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