express yourself

The 7th annual Protein Expressionmeeting (12–13 January 2004,San Diego, USA), which was organizedby the Cambridge Healthtech Institute(http://www.healthtech.com) as part ofits Protein Information Week, broughttogether over 200 researchers fromacademia and the biopharmaceuticalindustry to address the major issuesfacing protein expression.

Bill Studier of Brookhaven NationalLaboratories (http://www.bnl.gov/world) presented the keynote addresson his now ubiquitous T7 promotersystem, which is used for proteinexpression in Escherichia coli and formedthe basis for the development of theuniversally used pET system (Novagen;http://www.novagen.com). Almosttwenty years after its introduction, theT7 promoter system is still the system ofchoice for the majority of E. coliexpression. Studier also discussed howthe T7 promoter system has evolved,describing the development of anauto-induction media that wasspecifically tailored to the T7 promoterand was designed to meet the demandsof high-throughput expression.

Much of the conference was devotedto the problem of protein productionfor crystallography, with sessionsdiscussing protein expression forstructural determination and difficultto express proteins.

Protein expression for structuralgenomicsProtein expression has a direct impacton the successful outcome ofcrystallography. The overexpression ofproteins greatly facilitates purificationand subsequent protein production forcrystallography. Conversely, poor orinsoluble expression will impede the

purification of a homogeneous proteinsample, which is necessary forcrystallography. Although solubleexpression alone does not guaranteecrystallization, no or low expression,and thus the probable failure to obtainpure protein, is often cited as the mainreason that proteins are not crystallized.In the postgenomic era, the proteinproduction bottleneck in thecrystallographic determination ofprotein structure has been aggravatedby the abundance of genomicinformation that is available – there isno shortage of proteins to be expressedor structures to be determined.

Nowhere is this more clearlyillustrated than in structural genomics,where whole genomes are beingexpressed to provide structuralinformation that establishes functionand identifies novel folds. Proteinexpression on this scale requiresminiaturization and automation toachieve the levels of throughputrequired. At the forefront of such effortsis the Protein Structure Initiative (PSI;http://www.nigms.nih.gov/psi) researchprogram that is funded by the NationalInstitute of General Medical Sciences(NIGMS; http://www.nigms.nih.gov).Several researchers from the PSIconsortium described high-throughputprocesses for cloning, expression andpurification of protein for structuraldetermination purposes.

Ming Luo of the University ofAlabama at Birmingham (http://sgce.cbse.uab.edu) and Rosalind Kim ofLawrence Berkley National Laboratories(http://www.lbl.gov) described theirapproaches to the E. coli expression,purification and crystallization of proteinsfrom the Caenorhabditis elegans andMycobacterium tuberculosis genomes,

respectively. Although the combinationof miniaturization, automation andprocess design appears to haveaddressed the throughput problem,all of the PSI centers seem to be fallingshort in terms of actual output [1].

The greatest hindrance in thetransition from gene to crystal is seen asthe inability to express soluble protein.Scott Lesley of the Genomics Instituteof the Novartis Research Foundation(http://web.gnf.org/index.shtml) gavean impressive account of the high-throughput E. coli expression of theThermotoga maritima genome.Practising what was described as amulti-tiered approach, this processintroduces a focused effort andmethods for salvaging difficult toexpress proteins, including refoldingand alternative expression systems,which directly addresses the problemof low levels of expression. One of themost promising salvage techniquespresented involved using deuterium-exchange mass spectrometry to identifyregions of protein disorder, which couldpotentially interfere with crystallization.

Alternative expression systemsDmitry Vinarov of the University of Wisconsin at Madison(http://uwstructuralgenomics.org)discussed the use of a high-throughputwheat germ cell-free expression systemfor structural genomics. Wheat germcell-free protein synthesis, which wasdeveloped at Ehime University (Japan;http://www.ehime-u.ac.jp), has theadvantage of uncoupledtranscription–translation, producingslower and more favorable proteinfolding compared to other cell-freesystems typically using coupledtranscription–translation. Much of the

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Express yourselfStephen Chambers, Vertex Pharmaceuticals, 130 Waverly Street, Cambridge, MA 02139-4242, USA; e-mail: Stephen_Chambers@vpharm.com

interest in this system is because of theability to express soluble forms of someproteins that are not expressed in asoluble form in E. coli. In addition, theuse of cell-free expression systems isoften promoted because of the easewith which these systems produceisotopically labeled proteins for NMR structure determinations andseleno-methionine labeled proteinsfor phase determination in X-raycrystallography studies.

Unlike structural genomics,structure-based drug design focuseson the structural determination oftherapeutically relevant proteins andthus demands high output with littleopportunity for failure. In this differentenvironment, the emphasis is on highoutput. Stephen Chambers of VertexPharmaceuticals (http://www.vrtx.com)used E. coli and insect-cell high-throughput expression systems toidentify and deliver protein forstructure-based drug design. Parallelexpression of mammalian targets inE. coli and insect cells resulted in allproteins being expressed, with themajority produced as soluble proteinsin insect cells.

Philip Laible of Argonne NationalLaboratories (http://www.bio.anl.gov/laible) provided an excellent account ofthe fundamental problems encounteredwith membrane protein expression(not enough membrane) and made astrong case for membrane expression inRhodobacter, which have an abundanceof membrane invaginations comparedwith E. coli. Several bacterial membraneproteins were expressed in thisorganism, with the expression levels of many of the proteins reachingconcentrations of >10 mg/ml. Theseresults suggest that Rhodobacter is apromising system for the production ofmembrane proteins.

RefoldingGiven the relatively poor performanceof E. coli expression systems in

producing soluble protein, a numberof talks described alternative strategiesfor refolding protein from the inclusionbodies that are often produced.Paul Ramage of the Novartis Institutesfor Biomedical Research(http://www.nibr.novartis.com) gaveone of the most interesting talks.Following an overview of the state-of-the-art technology available in this area,Ramage went on to describe a novelapproach to the critical problem inrefolding – monitoring protein‘foldedness’. By coupling field-flowfractionation to light scattering,Ramage was able to develop andscreen a matrix of buffers to determinethe best conditions for proteinrefolding. Xinli Lin of ProteomTech(http://www.proteomtech-inc.com) hasa formidable track record of refoldinginsoluble proteins that have beenproduced in E. coli. Lin has adaptedhis past successes into an automatedsystem for the high-throughputscreening of protein-refoldingconditions. Given that estimates of thelevels of insoluble proteins expressed inE. coli ranged from 40% to 60%, eachof the refolding strategies presented atthe conference understandablygenerated considerable interest.

Protein fusionsAs a perennial favorite of theconference, protein fusions are oftencited as a universal remedy for insolubleprotein expression. A number of talkspresented data on the utility ofexpressing a protein as a fusion toimprove solubility, thereby facilitatingpurification. Pascal Braun of theInstitute of Proteomics, Harvard MedicalSchool (http://www.hip.harvard.edu)described the development of a high-throughput protein purification methodthat uses a number of the morecommonly used fusion-Tags, includingsix consecutive histidine residues (His),glutathione-S-transferase (GST),maltose-binding protein (MBP) and

calmodulin-binding peptide (CBP). Anassessment of the Tags (when used inE. coli ) identified GST- and MBP-Tags tobe superior to His- and CBP-Tags, withGST-Tags having a 50% success rate in purifying human proteins expressedin E. coli.

Tauseef Butt of LifeSensors(http://www.lifesensors.com) andPhilip Bryan of the Center forAdvanced Research in Biotechnology(http://www.carb.nist.gov) describedsuccessful fusions with small ubiquitin-like modifier (SUMO) and asubtilisin-proregion fusion, respectively.

Something of a clash of cultures (anduses) was evident with one speaker.Harry Meade of GTC Biotherapeutics(http://www.transgenics.com) thoughthe had gone back in time 20 years.Meade described the successfulexpression of a number of complexprotein therapeutics, including vaccines,hormones, antibodies and solublereceptors, in transgenics. With noproblem expressing an abundance ofsoluble protein in herds of lactatingtransgenic goats, Meade was somewhatbemused as he heard those involved instructural genomics wrestle with thedifficulties of producing soluble proteinin E. coli. This problem is one that theprotein therapeutics arm of thebiotechnology industry resolved longago in favor of using eukaryoticsystems, which are more adept atmanaging the complexities of refoldingand producing soluble protein. Theargument for using an E. coli expressionsystem is that this system combines therobust expression and simplicity ofprotein production, making it the idealexpression system to deliver proteinfor crystallography. In addition, E. colilacks the post-translational machineryof eukaryotic systems and, therefore, is more suited to producing ahomogeneous protein sample. This isthe key to the devotion of thecrystallographer to using E. coli as anexpression system. Furthermore, the

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value given to homologous proteindrives the use of E. coli for structuredetermination purposes.

Automation The automation of protein expressionenables researchers to cope with theproliferation of protein expressionstrategies available and a number ofpresentations described equipmentthat would perform this function.Grant Cameron of NextGen Sciences(http://www.nextgensciences.com)described the Expressionfactory™, aninstrument that automates cloning,expression and purification within asingle platform. When combined witha powerful proprietary informationmanagement system, differentcombinations of expression vectorsand hosts can be used to expressprotein, thus enabling the parallelexploration of different growth andpurification strategies. Nina Forsbergof Amersham Biosciences

(http://www.amershambiosciences.com)presented recent developments in theAKTA-3D purification system, which iscapable of automating affinitychromatography, gel-filtration and ion-exchange chromatography. The AKTA-3Dsystem can automatically purify sixdifferent His- or GST-tagged proteins to>95% purity, with yields of 50 mg.Throughput can be maximized by usingfour AKTA modules in parallel to enablethe purification of up to 16 proteins ina single run.

Concluding remarksThe manufacture of recombinantprotein therapeutics and the productionof target proteins for drug screeninghave dominated previous proteinexpression conferences but, with thedevelopment of structural genomicsand proteomics, that emphasis ischanging. The challenge now ofproviding protein content for structuralstudies and protein interactions has

altered the character of proteinexpression and, as a consequence, theconference. This change is reflected inthe content of many of the talks, whichnow encompass employing automationin the protein expression process, thusdictating more universal and genericstrategies for expression, in comparisonto the highly customized strategies thatwere previously used.

Although the PSI has been criticized fornot immediately delivering the wealth ofnew structures once thought possible, ithas made an impact on the developmentof new technologies for proteinexpression, production and analysis. Justas the Human Genome project aidedthe development of DNA technology,the PSI is advancing the technologiesthat underpin structural genomics, andin particular protein expression.

Reference1 Service, R.F. (2002) Tapping DNA for structures

produces a trickle. Science 298, 948–950

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