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High-throughput biology has been pioneered by genomicsthrough the application of robotics to expedite DNA-sequencing projects. Advances in high-throughput proteinmethods are needed to drive the protein production line forhigh-throughput structural and functional analysis of newlydiscovered genes. This will require the development andapplication of a variety of recombinant-protein expressionsystems to produce the diversity of proteins from both humansand model organisms.

AddressesBiology and Biotechnology Research Program, Lawrence LivermoreNational Laboratory, Livermore, California 94550, USA*e-mail: albala1@llnl.gov

Current Opinion in Chemical Biology 2001, 6:102–105

1367-5931/01/$ — see front matter© 2001 Elsevier Science Ltd. All rights reserved.

Published online 19 December 2001

AbbreviationI.M.A.G.E. integrated molecular analysis of genomes and their expression

Introduction The past year has seen the completed sequence of thehuman genome [1••,2••] added to the catalogue ofsequenced genomes from other species, including severalmicrobes [3,4], fly [5] and worm [6]. The key applicationfor newly decoded genetic information is the identificationof open reading frames that translate into the thousands ofproteins that enable life. Understanding the biological relevance of newly discovered genes and the proteinsencoded therein is the impetus for the emerging field offunctional genomics. Development of methodologies toproduce hundreds or thousands of proteins in parallel isessential to obtain the quantity of proteins necessary forhigh-throughput biological, biochemical or pharmaceuticalanalysis. Application of high-throughput protein-productionstrategies will provide the foundation for the analysis ofnewly identified genes and genomes necessary to deciphercomplex pathways and interactions within the cell.Hopefully, this will lead to the understanding not only of gene function but also dysfunction, and the causality of disease.

Some of the key features in developing strategies for high-throughput protein analyses are parallelization, minia-turization and automation [7,8]. High-throughput proteinproduction must ideally encompass a series of steps thatcan be performed on many genes simultaneously, in amulti-well format, using robotic manipulation, and requir-ing minimal human intervention. Each step must be robust,reproducible and, critically, protein-independent. Currenthigh-throughput functional analysis procedures dictate thatproteins be produced and purified in microgram to

milligram quantities, at a rate of hundreds to thousands ofproteins per day. To achieve this, purification proceduresshould require a minimal number of steps and result in proteins that are correctly folded and non-labile, with fewdetectable impurities and, most importantly, functional. Asproteins are a diverse population of biomolecules, each withunique biochemical properties, development of genericprotocols are required for the purification of as many different proteins as possible to maintain the production-line scheme. Having purified proteins in-hand will facilitatefunctional and structural genomics efforts for novel discoverytowards diagnostics and therapeutic applications.

Applications for high-throughput proteinproductionThe primary scientific application driving the protein production line comes from the structural biology community. In the emerging field of structural proteomics,proteins of high purity and concentration are needed for high-throughput NMR and X-ray crystallographic structure determination, both to understand protein function and for rational drug design [9,10••,11]. The goalof structural proteomics is to obtain the three-dimensionalstructure of all proteins as well as the cataloging of all existing protein folds to gain a functional understanding ofunknown proteins by structural homology [11]. Currentefforts are focused on the more accessible proteins readilygenerated in Escherichia coli, which provide effective incorporation of selenomethione for X-ray crystallographyor 15N/13C for NMR [12].

A second main application requiring large numbers of proteins is the design of ‘protein chips’. Protein chips provide a means to perform parallelized analyses with theability to identify each protein by its addressable positionon the array, to measure protein expression, protein–pro-tein interactions or other protein functions. Array-basedproteomics will take many forms as different types of biological materials, primarily proteins, peptides and anti-bodies, can be analyzed using a microarray, microfluidic- orbead-based format (for reviews see [13–15]). Additionally,the successful generation of thousands of proteins will provide the basis for the production of thousands of highlyspecific antibodies or antibody mimics for the design andmanufacture of antibody chips for use in molecular diagnostics and therapeutics [15,16•].

Current high-throughput protein-productiontechniquesProkaryotic expressionThe most common method for obtaining large numbers of recombinant proteins is to generate them in E. coli.Recombinant protein technology was developed in E. coliand it remains the most prevalent system for protein expression

Accelerating code to function: sizing up the protein production lineMichele Gilbert and Joanna S Albala*

Accelarating code to function: sizing up the protein production line Gilbert and Albala 103

because of its robust production, low cost and ease of use.Several labs have successfully produced and purified largenumbers of proteins using high-throughput strategies inE. coli, either by recombinant or in vitro means [10••,11,17–19].An excellent example comes from examination of the pro-teome of Methanobacterium thermo-autotrophicum, from which424 non-membrane proteins were cloned by PCR and produced as candidates for structural analyses. Of these,approximately 80% could be expressed in E. coli and 100soluble proteins were purified resulting in the successfuldetermination of 10 structures [10••]. However, it is note-worthy that this study concluded that insufficient proteinexpression and solubility accounted for almost 60% of theunproduced proteins. Thus, additional strategies beyondE. coli are needed to clone, express and purify proteins forstructural genomics applications.

A different type of proteomics approach has been taken toselect and produce proteins after initial library screening.In one study to produce proteins for antibody productionand subcellular localization studies, 55 unknown cDNAclones were randomly selected from a mouse testis cDNAlibrary, amplified using a PCR-based strategy and clonedinto E. coli. This resulted in 49 purified proteins producedin the range of 1–10 mg, sufficient for immunization [17].A similar strategy has been employed by Bussow et al. [19],who constructed a human brain cDNA library in E. coli,and arrayed it onto polyvinylidene difluoride (PVDF) filtersfor analysis of protein expression using an anti-epitope tagantibody. 96 clones were randomly selected from thislibrary and subsequently used to produce the recombinanthuman proteins in a high-throughput microtiter-plate format [20].

Automation is key to providing the throughput needed forproteomic studies involving tens of thousands of proteins.Many of the methodologies of protein production lendthemselves to robotic manipulation because of the repeti-tive nature of procedures such as plasmid isolation, PCR,DNA quantitation, cell culture and affinity purification.Moreover, automated procedures minimize human errorand decrease costly personnel numbers. In one study toidentify the expression of recombinant soluble protein,small volumes of bacterial cultures (1–2 ml) were grown indeep-well microtiter plates and lysed in specialized96-well plates. The soluble fraction was then separatedfrom the insoluble inclusion bodies by vacuum filtrationand subsequently transferred to a membrane support fordirect detection of expressed protein [21].

Several aspects of high-throughput protein production canbe accomplished using robotics, as has been applied togenomics efforts such as aspiration, dispensing, pipetting,vacuum filtration and plate-shaking. Moreover, despite theobvious difficulties of cost and space, large-scale proteinpurification can be automated and an innovative, industrial-scale system has been devised for processing 96 parallel,65–70 ml bacterial cultures. These extensive custom

robotics also incorporate centrifugation and sonicationcapabilities required for purification at these larger volumes and the system is able to process 96–192 proteinsper day with average yields of 10 mg of purified proteinthat is typically 80–90% pure [22].

Beyond bacteriaAlthough E. coli-based recombinant protein production hasremained fundamental to many academic and commerciallabs, there are some inherent disadvantages to using aprokaryotic system for heterologous protein production,such as formation of inclusion bodies, misfolding of proteins and lack of post-translational modifications. Theuse of E. coli-based recombinant expression systems oftenresults in the production of insoluble inclusion bodies,requiring significant efforts to resolubilize if no usable pro-tein is generated from the initial production path [11].Historically, fusion of the heterologous protein to a partnercan reduce inclusion body formation as well as provide ameans for affinity purification. In order to avoid improperlyfolded protein (often the cause of inclusion-body forma-tion), many labs have utilized chaperones and foldases [23]or factorial screening methods [24]. For high-throughputexpression systems, the need to successfully refold proteins is unlikely to be automatable and, furthermore, isusually protein-dependent.

A critical issue when using prokaryotic systems is the lackof glycosylation and phosphorylation of the resultantrecombinant proteins. Many proteins require these post-translational modifications to function as well as to foldcorrectly [25–27]. Enzymatic modification to provide theserequired post-translational modifications can be appliedafter purification, but these processes are costly and canresult in large variation in the modifications [27].

Eukaryotic expression systemsIt may be impractical to expect to produce human proteins,of diagnostic and therapeutic interest, in prokaryotes. Toovercome many of the limitations arising from prokaryoticexpression, several eukaryotic systems have been devel-oped utilizing either yeast, insect or mammalian cells forhost expression. These eukaryotic systems all have thecapability to properly phosphorylate and glycosylaterecombinant proteins to varying degrees [26,28]. Dual usemethods for recombinant expression of prokaryotic andeukaryotic systems have also been devised to expandrecombinant protein production capability [29]. UsingSaccharomyces cerevisiae for heterologous protein produc-tion, 2000 colonies from a human fetal brain cDNAexpression library were robotically arrayed into 384-wellplates. 96 clones were randomly selected, cloned by PCRand subjected to sequence analysis and protein expression.The results showed that 58 of the 96 clones were in-framewith the selection tag and the resulting recombinant proteins were detected using high-density protein arrayswith an epitope-specific antibody before being producedin large quantities for functional analyses [30].

104 Proteomics and genomics

In pioneering work by Zhu et al. [31••], all the open reading frames from the genome of S. cerevisiae were independently cloned into a yeast expression systemresulting in glutathione-S-transferase-fusion proteinsderived from 5800 yeast cDNAs. The proteins were purified in a non-automated fashion in a 96-well formatand spotted onto glass slides for functional analysis. In anexemplary experiment, 150 protein–lipid interactions wereidentified by this proteomic analysis. Although recombi-nant expression in yeast has been applied successfully tohigh-throughput paradigms, yeast cannot provide all therequirements for heterologous protein production such ascorrect recognition of signal peptide sequences [25]. Thereis an example of a 96-well format that has been devised forproduction of human proteins in COS cells [18]; however,recombinant expression in mammalian cells suffers fromreduced expression by comparison to other host systems.Although it is evident that no single expression system willsatisfactorily produce every protein introduced, use ofeukaryotic expression systems will be needed to producemore complex human proteins with extensive post-transla-tional modifications as well as more traditionally difficultto produce proteins, such as membrane-bound proteins. Itshould be noted that several human membrane proteinshave been made successfully using baculovirus as themeans for recombinant expression, as insect cells are ableto utilize the native signal sequence of the protein to beproduced [28].

Protein production in baculovirusThe baculovirus system lends itself well to high-through-put protein production, and recombinant expression inbaculovirus has been engineered to produce a wide rangeof foreign proteins in milligram quantities. Insect cellsgrow readily in suspension without the need for carbondioxide, induction or specific temperature conditions.Although insect cell cultures are typically not as hardy asbacterial cultures, they are more durable than mammaliancells for high-throughput applications.

We have developed a strategy for high-throughput proteinexpression using baculovirus employing I.M.A.G.E. (inte-grated molecular analysis of genomes and their expression)cDNAs [32] for the production of human proteins in aeukaryotic environment [33]. Our strategy for high-throughput protein expression and purification utilizes amicrotiter format for all manipulations, amenable toautomation. A prototype plate containing 88 I.M.A.G.E.cDNAs and eight controls was rearrayed from 384-wellmaster plates into the 96-well format. Recombinant baculoviruses were produced using a PCR-based methodthat eliminates plaque purification. The resulting PCRproducts were purified and incorporated into recombinantbaculovirus in the 96-well format using a standard transfec-tion protocol. Cells infected with amplified recombinantvirus were harvested after 72 hours for protein expression.The expressed proteins incorporate an amino-terminal epitope (Glu–Glu) tag to facilitate purification and analysis.

A miniaturized 96-well cell culture system has beendesigned for the culturing of small volumes (1–2 ml) ofinsect cells. Lysates prepared from insect cells infectedwith recombinant virus were adsorbed onto animmunoaffinity matrix in a microtiter format and recombi-nant protein was eluted by competition with syntheticpeptide. Western blot analysis using an antibody to theepitope tag demonstrated 42% soluble recombinant pro-tein was produced from the protoplate. Arrayed, purifiedrecombinant proteins can subsequently be used for high-throughput functional analysis or high-throughputcrystallization trials for structural studies. Although there isevidence that proteins produced using baculovirus forrecombinant expression can incorporate selenomethioninefor X-ray crystallography studies [34], this has yet to bedemonstrated in a parallelized process.

ConclusionsAlthough high-throughput DNA sequencing and analysishave been automated and optimized, the protein produc-tion line is still under development. Many of the roboticsand automation systems that revolutionized DNAsequencing are being applied to the expression and purifi-cation of various proteomes. Current high-throughputprotein production employs primarily E. coli but mustinevitably progress to the utilization of eukaryotic systemsto generate those proteins that are traditionally difficult toexpress and purify. We feel that the baculovirus expressionsystem will be critical to heterologous, high-throughputprotein production beyond bacterial systems. The rise ofnew systems and expansion of existing strategies for theproduction of soluble proteins will certainly propel func-tional genomics and proteomics well into the 21st century.

AcknowledgementsThis work was performed under the auspices of the US Department of Energyby the University of California, Lawrence Livermore National Laboratoryunder Contract No. W-7405-Eng-48. The authors would like to thank Ian RMcConnell and Christa Prange for their critical review of the manuscript.

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