Protein Expression and Purification22,159–164(2001)160SCOTT A.LESLEYTABLE1Genomic versus Proteomic TechnologiesGenomic technologies(DNA)Proteomic technologies(protein) Identification Determined experimentally,bioinformatics Predicted from genomic informationFunction1-dimensional information storage3-dimensional organization of chemicalfunctionalitiesBuilding blocks4bases20ϩamino acidsDetection sensitivity PCR amplification techniques Direct detection methodsSynthetic approaches Cheap and efficient oligonucleotide synthesis Limited capacity of peptide synthesismethods combined with PCRSequence determination500–700bases common by automated sequencing Direct sequencing difficult,mass spectrometry Purification Generic methods Generic methods require modification of proteinthrough gene fusionAnalysis methods Typically employ enzymes,hybridization Chemical,biophysical,biochemicaland mutagenesis),interactions between proteins(two-are of primary interest and typically are expressed in hybrid),and global protein changes(2D gels and LC–a bacterial host.Often this approach leads to problems MS).Purified protein is often required in these studies associated with expression levels and proper folding of and defines the outputs of any parallel expression and the protein of interest.Flexibility in expression options purification process.is a key parameter.Pichia or baculovirus expressionsystems can offer effective alternatives to bacterial sys-tems.Each expression scenario requires a specific vec-GENE CLONING FOR EXPRESSIONtor.Recloning cDNAs into each of these specific vectors Determining gene function through genomics typi-is extremely labor-intensive.Recombinatorial cloning cally starts from a query of a database.Sequence infor-methods provide an opportunity to minimize the effort mation for the3.9billion bases of sequence from the required for alternate expression.human genome is now available(1,2).Access and inter-Two systems are commonly used for recombinatorial pretation of this information often require sophisticated cloning and shown in Fig.1.The cre–lox recombination bioinformatics software outside the scope of this dis-system described by Elledge utilizes a single recombina-cussion.Public archives such as the Unigene Data-tion to introduce the gene of interest into a recipient base(/)or TIGR(http://vector(3).This is an in vitro reaction combined with a /)provide bioinformatic access to many in-genetic selection for the recombinant vector.In this way teresting plete genomic sequence infor-the gene of interest is cloned once into a donor vector mation is now available through Celera(http://and can then be moved into any of a number of recipient /index.cfm).Future annotation of fullplasmids for expression in different hosts or to utilize sequence information will greatly expand the access todifferent purification tags.A similar system utilizes full-length cDNA sequences.The first requirement is converting this genomic in-lambda Int/Xis/IHF recombination at att sites(4)to formation into an actual cDNA clone of that gene.Am-achieve transfer of open reading frames(ORFs).This plification of full-length cDNAs via PCR is the typical system has the advantage of a precise ORF transfer to first step.Both reverse transcriptase and amplification the expression vector rather than the cointegrant vector polymerases typically are lacking in proofreading activ-product of the cre–lox system.With either system,the ity.Care must be taken to limit the number of steps of primary limitation is that translational fusion of the amplification and to use proofreading enzymes where recombination sites is typically required to maintain possible to minimize the probability of introducing un-the flexibility and utility of the recombination method wanted mutations.Tissue selection for cDNA librariesfor expression.In those cases,such as crystallography, also is an important consideration for attempting towhere translational fusions are potentially detrimental isolate genes as they must be expressed within thatto the protein,a conventional cloning approach is library source for successful amplification.This infor-more appropriate.mation is often obtained from cDNA or oligonucleotideRegardless of the cloning method,parallel expression expression arrays.and purification requires utilization of purification Amplified gene products are cloned into appropriatetags.Many options exist for this purpose.A comprehen-vectors for expression.Depending on the source of thesive review is beyond the scope of this article.A list of gene,the host,and the end use of the protein,manydifferent vectors may be appropriate.Eukaryotic genes some commonly used tags is shown in Table2.By farHIGH-THROUGHPUT PROTEOMICS161FIG.1.Strategies of recombinatorial cloning.Individual cDNAs are cloned into a donor vector that can then be recombined into any number of recipient vectors through recombinatorial cloning.One option is to form a cointegrant plasmid through Cre-mediated recombination across a lox site.In the second scenario,a cDNA is flanked by phage lambda att sites which direct recombination into an expression vector through the use of the INT/XIS/IHF proteins.In these ways,a single donor clone can easily be transferred into any number of recipient vectors. the most common fusion is the histidine tag for purifica-analysis studies into small-scale for analysis of expres-tion on metal-chelate resins.This tag provides a sub-sion levels and properties and into large-scale for use stantial purification handle while being relatively un-with many of the proteomic applications.obtrusive as a fusion partner.Beyond purification,Small-scale expression is most useful for identifying translational fusions often provide a means to enhance those clones which express recombinant protein to high expression.The larger fusion tags such as thioredoxin levels and for evaluating the folding state of the protein. and GST often are superior in this respect.Crude expression testing is typically done by simpleSDS–PAGE analysis of whole cells.Evaluation of thefolding state is typically done by centrifugal fraction-RECOMBINANT EXPRESSION OF PROTEINation of a lysate,requiring a more gentle lysis proce-dure.Several lysozyme and mild detergent methods are Bacterial expression is most common for recombinantcommercially available for this purpose.proteins because of its ease of use and the high levelsof protein obtained.It is useful to divide expression For large-scale production for many applications,TABLE2Common Purification TagsBasis of purification Elution Reference Small tagsHistidine tag Metal affinity resin Imidizole(5)S-tag Interaction with S-protein Temperature(6) Calmodulin-binding protein Interaction with Calmodulin Calcium(7) Large tagsGlutathione S-transferase Glutathione agarose Reduced glutathione(8) Thioredoxin Phenylarsine oxide resin-Mercaptoethanol(9) Biotinylation domain Monomeric avidin resin Biotin(10) Maltose binding protein Amylose resin Maltose(11) Chitin binding protein/intein Chitin affinity Thiol(12)162SCOTT A.LESLEYtens of milligrams of protein typically are required.PURIFICATION STRATEGYEven with common bacterial expression levels,500–Proteins are highly diverse in their properties mak-1000ml of culture typically is required to provide these ing generic methods of purification difficult.Purifica-amounts.While such methods are commonplace in labo-tion tags such as described in Table2are a typical ratories,systems for parallel processing large numbers solution for purifying proteins in parallel.His-tag fu-of cultures at this level are not commercially available.sions are very common and provide a single-step chro-By developing instrumentation and optimizing media matographic purification that yields protein of suffi-and aeration conditions for high-density cell growth,cient purity for most applications.In addition,the his-our laboratory can parallel process96cultures at this tag sequence requires the addition of only six amino scale.Optical density(OD)values at600nm can reach acids to the recombinant protein,reducing the likeli-a value of40with logarithmic growth through at least hood that such a fusion will adversely affect gene func-30OD units.These cell densities allow us to producetion.A typical purification strategy is outlined in Fig.2. sufficient cell mass with65-ml culture volumes to yieldtens of milligrams of recombinant protein,sufficient for PURIFICATION AUTOMATIONmost applications.Such instrumentation is not com-Parallel processing typically involves instrumenta-monly available,but common shaking incubators cantion for automation.Lysis methods such as sonication or substitute with larger volumes.using a French press are not simple automation tasks. Recombinant expression of proteins is achievedLikewise,centrifugation is not easily integrated into through induction of a strong promoter system.Manyautomation due to problems of locating and indexing options exist in this regard including tac,T7,lambdathe rotor position.Automation of this process typically P L,and ara B promoters.It is important for parallelinvolves protocol modifications.This can easily be processing that growth and induction characteristicsachieved in small-scale methods.are consistent.For this reason,it is important to retainOn small-scale,parallel processing usually involves tight repression of expression and have a simple induc-use of a96-well plate format.Lysis is typically achieved tion procedure for high-level expression.For T7sys-using a combination of lysozyme and freeze–thaw cy-tems,the lac operator and T7lysozyme(pLysS)provide cles.Phage lysozymes are more effective than hen egg an extra level of repression.The arabinose promoter is white lysozyme for this purpose and can be combined tightly repressed in the absence of inducer and is our with nucleases to reduce viscosity and facilitate re-preferred system for parallel growth.With all of the moval of cell debris at the low g forces commonly used promoters listed,recombinant expression levels of10–with microtiter plates.Alternatively,nonionic deter-50%of total cell protein are common.gents can be employed for nondenaturing lysis.FIG.2.Generalized purification strategy of recombinant fusion protein.A common purification strategy is shown here.Proteins are purified from fermentation cultures by affinity purification.Isolated cell pellets are resuspended in an appropriate lysis buffer and disrupted by high-intensity sonication.Cell walls and insoluble debris are pelleted by centrifugation and the soluble supernatant containing the recombinant fusion protein is applied to chromatography resin containing an immobilized metal for affinity purification.Fractions containing the recombinant protein can be used directly or further purified using conventional chromatographic techniques.HIGH-THROUGHPUT PROTEOMICS163TABLE3Robotic Systems with Capabilities Adaptable to ProteinPurificationManu-facturer Instrument WebsiteQiagen BioRobot3000/Tomtec Quadra96/Matrix PlateMate /Hamilton Microlab4200/Beckman Biomek2000/Packard PlateTrak /index2.htmGilson Nebula215/index.htmlRobotic systems for nucleic acid purification are rela-tively commonplace and have recently been adapted forprotein purification.The Qiagen BioRobot3000per-forms multiple functions relevant to protein purifica-tion.It provides aspirate,dispense,pipet,vacuum fil-tration,and plate-shaking functions on a relativelycompact platform.These functions can be adapted to FIG.4.SDS–PAGE analysis of purified protein.Metal affinity chro-perform cell lysis and chromatography steps from1–2matography yields highly purified protein from a single chromato-graphic separation.This gel shows typical yields and purity obtained ml of bacterial culture.Specialized96-well plates clearfrom parallel purification using an automated purification system. cell debris via vacuum filtration and are also used toSuch proteins have been incorporated directly in successful crystalli-retain resin for chromatographic separations.The Wal-zation trials.Ten-microliter samples of12ml protein eluates from Ni-lac Quadra96also has most of these capacities and resin were separated by10%SDS–PAGE.Samples are recombinant can parallel process96or384samples.Both of thesefusions of thioredoxin to human proteins as indicated by accessionnumber.systems have been used with success in our laboratoryfor small-scale protein purification of proteins in micro-titer plates.Table3lists some robotic systems that maybe applied to small-scale protein purification.providing the throughput needed for proteomic studies Despite the difficulties,large-scale protein purifica-involving tens of thousands of proteins.We are cur-tion also can be automated.In our laboratory we simul-rently able to process approximately96–192proteins taneously process96bacterial cultures of65–70ml.per day with this system with average yields of around Instrumentation for processing96parallel cultures at10mg of purified protein.Affinity purification results that scale required development of custom roboticsin recombinant protein that is typically80–90%pure shown in Fig.3.These robotics incorporate liquid aspi-(see Fig.4)which is sufficient for most applications. rate and dispense,centrifugation,and sonication capa-Subsequent purification is sometimes necessary,for ex-bilities required for purification.Automation is key to ample,in protein crystallography,and is achieved usingFIG.3.Protein purification automation.Custom robotics for performing the purification strategy outlined in Fig.2are shown.(a)The instrument has capacity for automated liquid aspiration and dispensing,sonication,centrifugation,and fractionation.Ninety-six cultures are processed in parallel,giving up to10–50mg of purified protein per culture.(b)Expanded view of aspirate/dispense/sonicate head accessing rotor.164SCOTT A.LESLEYREFERENCESstandard ion-exchange and size-exclusion chromatog-raphy.Automation of these methods is relatively1.Venter,J.C.et al.(2001)The sequence of the human genome. straightforward employing standard FPLC and au-Science291,1304–1351.tosampler instrumentation.2.International Human Genome Sequencing Consortium(2001)Initial sequencing and analysis of the human genome.Nature SUMMARY409,860–921.3.Liu,Q.,Li,M.Z.,Leibham,D.,Cortez,D.,and Elledge,S.J. 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Knuth,Loren Miraglia,and Jeremiah Gilmore for their contributionsto the high-throughput cloning and expression efforts.He also recog- A.,Pelletier J.J.,Paulus H.,and Xu M.Q.(1997)Single-column nizes Bob Downs,Mark Weselak,Andy Meyer,and Jim Mainquistpurification of free recombinant proteins using a self-cleavable and the rest of the GNF engineering staff for their contributions to affinity tag derived from a protein splicing element.Gene192, the custom robotics that make this effort possible.271–281.。
