Design of high-throughput methods of protein production for structural biology

Abstract

High-throughput protein expression and purification has a central, pivotal role in structural genomics. In fact, crystallographic-quality protein production on the scale required to generate tens to hundreds of different proteins per day will probably be the greatest obstacle for the conversion of protein structure determination to a high-throughput format. High-throughput efforts in structural biology place unique restrictions on protein expression and purification. First, the majority of gene constructs must be expressed in a synchronous fashion. Second, the purification protocol applied to the majority of expressed proteins must be as similar as possible and produce the very high-quality material that is needed for structural studies. The first requirement can be addressed by employing either large or small N-terminal expression tags, and the second hurdle can be circumvented with the use of affinity purification tags. However, the drawback in incorporating affinity tags in crystallization studies is that in many cases these tags introduce flexible portions to the protein of interest that are not conducive to crystallization or lead to various forms of microheterogeneity. Protease cleavage sites allow removal of these flexible tag regions, but conditions often have to be optimized for each reaction, requiring fine-tuned processing to be incorporated in a high-throughput environment. Given the above caveats on current technology, however, affinity-tag systems are still the most useful to date given the restrictions placed on high-throughput methods. Several groups and commercial companies are currently designing high-throughput protein production, in particular, systems based on Escherichia coli are described in this review. In addition, other systems are being investigated, including in vitro expression systems [1Kigawa T Yabuki T Yokoyama S Large-scale protein preparation using the cell-free synthesis.Tanpakushitsu Kakusan Koso. 1999; 44: 598-605PubMed Google Scholar, 2Kigawa T Yokoyama S et al.Cell-free production and stable-isotope labeling of milligram quantities of proteins.FEBS Lett. 1999; 442: 15-19Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar], methods that employ baculovirus infection of insect cells with a peptide tag for antipeptide monoclonal antibody purification [3Albala J.S Humphery-Smith I Array-based proteomics: high-throughput expression and purification of IMAGE consortium cDNA clones.Curr. Opin. Mol. Ther. 1999; 1: 680-684PubMed Google Scholar], and yeast-based expression using a single tag [4Schuster M Werner G et al.Protein expression strategies for identification of novel target proteins.J. Biomol. Screening. 2000; 5: 89-97Crossref PubMed Scopus (15) Google Scholar] or dual tags [5Rigaut G Shevchenko A Rutz B Wilm M Mann M Seraphin B A generic protein purification method for protein complex characterization and proteome exploration.Nat. Biotechnol. 1999; 17: 1030-1032Crossref PubMed Scopus (2227) Google Scholar]. A distinct advantage of in vitro expression systems is the facilitation of selenomethionine (SeMet) incorporation or 15N labeling. Scale-up has been a problem with this technology, however, and is currently being addressed [1Kigawa T Yabuki T Yokoyama S Large-scale protein preparation using the cell-free synthesis.Tanpakushitsu Kakusan Koso. 1999; 44: 598-605PubMed Google Scholar, 2Kigawa T Yokoyama S et al.Cell-free production and stable-isotope labeling of milligram quantities of proteins.FEBS Lett. 1999; 442: 15-19Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar]. Because all of these systems are still in the early stages of development, data collection in a systematic and comprehensive manner will be necessary to arrive at the most efficient and cost-effective solution for high-throughput protein expression and purification. The parallel production of protein targets, expressed under a range of conditions with a variety of affinity tags, will result in a number of successful conditions for protein production, along with many unsuccessful conditions. By analyzing which conditions produce viable samples for a given type of protein and which conditions do not work, a large knowledge base will be produced. This knowledge base will provide the foundation for a statistically relevant method of predicting effective expression conditions for protein samples with similar physical and biochemical properties. As this knowledge base grows in volume, the predictive power should improve, resulting in a larger number of successes and an exponential growth of the knowledge base itself. Through this iterative process, trends in protein production will become apparent, and the speed and efficiency of high-throughput protein production will be greatly enhanced. Each aspect from cloning to protein quality control is discussed here with an emphasis towards high-throughput methods and collection of success/failure data in a systematic manner. Recombinant protein yield and solubility are highly dependent on the specific protein sequence, as well as on the vector, host cell, and culture conditions used. For optimal efficiency, various combinations should be simultaneously screened to determine the conditions that yield the ‘best’ protein, for example, choosing five different expression clones for each protein of interest (see Figure 1). Cloning using restriction enzymes typically cannot be used for high-throughput approaches, owing to the complication of selecting compatible and appropriate restriction enzymes for each cloning procedure. Additionally, multiple steps of experimental refinement and treatment must be performed using the restriction enzyme(s) of interest. High-throughput cloning therefore requires procedures based on the polymerase chain reaction (PCR). A first step involves the design of specific gene PCR amplification primers, followed by screening of potential PCR-amplified clones for proper insert orientation. Sequence analysis of positive clones must be performed, to confirm that a proper reading frame has been obtained and that no PCR-introduced errors are present. This step is followed by a final archiving of selected plasmid DNA samples. Automated systems are available for colony picking, gridding, and microarraying (e.g. the ‘Q Pix’ system sold by Genetix [http://www.genetix.co.uk] and the Gene Suite™ available from GeneMachines [http://www.genemachines.com]) and also for sequencing (e.g. fluorescence-based systems available from Perkin Elmer Biosystems, Amersham Pharmacia Biotech, and Visible Genetics). To generate expression vector clones, cloning systems such as the Invitrogen Echo™ system [6Liu Q Li M.Z Leibham D Cortez D Elledge S.J The univector plasmid–fusion system, a method for rapid construction of recombinant DNA without restriction enzymes.Curr. Biol. 1998; 8: 1300-1309Abstract Full Text Full Text PDF PubMed Google Scholar], the Gibco/Life Technologies Gateway™ system, or the Novagen pTriEx-1 cloning system [7Novy R Yaeger K Monsma S Scott M pTriEx-1 multisystem vector for protein expression in E. coli, mammalian, and insect cells.inNOVAtions. 1999; 10: 1-5Google Scholar] may be advantageous. These generic processes streamline the expression cloning process by alleviating costly, time-consuming recloning steps and avoiding the use of restriction enzymes in the cloning and subcloning process. For example, the Invitrogen system inserts the PCR-amplified gene fragment into a TOPO™ ‘donor’ vector using a topoisomerase-I-adapted plasmid. CRE recombinase is then used in a second step to introduce the correct cloned sequence into a loxP-adapted recipient vector (E. coli, insect, yeast, and mammalian choices are all available) for subsequent protein expression studies. High-throughput approaches will rely upon both prokaryotic and eukaryotic hosts. Bacterial expression systems are advantageous for a variety of reasons, most notably that protein overexpression is usually obtained without any post-translational modification heterogeneity. In addition, E. coli protein expression is cheaper and faster than eukaryotic systems. In comparison, more expensive and slower eukaryotic systems will be necessary for the expression of some subsets of proteins, especially those that require post-translational modifications for proper folding and activity. Currently, many companies have available a variety of vector/host expression systems (see the Supplementary material section). Numerous heterologous gene expression systems are available (see the Supplementary material section), and additional variants can be constructed using combinations of strong promoters and tight regulators (as listed in Table 1) along with the proper transcription initiation and translation signals. These systems produce recombinant gene products in an efficient and regulatable manner [8Makrides S.C Strategies for achieving high-level expression of genes in Escherichia coli.Microbiol. Rev. 1996; 60: 512-538Crossref PubMed Google Scholar, 9Fernandez J.M Hoeffler J.P Gene Expression Systems: Using Nature for the Art of Expression. Academic Press, San Diego, CA1999Google Scholar]. Cost-effective E. coli expression, using T7 RNA polymerase plus the T7 promoter [10Studier F.W Moffatt B.A Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes.J. Mol. Biol. 1986; 189: 113-130Crossref PubMed Scopus (4699) Google Scholar, 11Studier F.W Rosenberg A.H Dunn J.J Dubendorff J.W Use of T7 RNA polymerase to direct expression of cloned genes.Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (5918) Google Scholar] with induction for high-yield recombinant protein overexpression, appears to be appropriate for ‘first pass’ efforts.Table 1Control elements used for E. coli recombinant protein expression.PromoterRegulationInducerLevel of expressionCommentT7 bacteriophagelacIqIPTGVery highUtilizes T7 RNA polymerase. High-level inducible overexpression commonly obtained.T7lac system for tight control of inductionneeded for more toxic clones.Expensive induction.trc (hybrid) E. colilacI, lacIqIPTGModerately highLower level expression versus T7 systems, but high-level, regulated expression still possible.Expensive induction.pL (λ)λcIts857Temperature shift to 42°CModerately highTemperature-sensitive host required. Less likelihood of ‘leaky’ uninduced expression 80Shatzman, A.R., Gross, M.S. & Rosenberg, M. (1997). Expression using vectors with phage λ regulatory sequences. In Current Protocols in Molecular Biology. pp. 16.3.8–16.3.11, John Wiley and Sons, Inc, New York, NY.Google Scholar.araBADaraCl-ArabinoseVariable, from high to low levelCan fine-tune expression levels in a dose-dependent manner (tight regulation possible).Inexpensive inducer. Open table in a new tab As previously mentioned, there is an important advantage in using tags in high-throughput protein expression and purification efforts, so that all proteins will have a generic ‘handle’. Several N-terminal expression tags are available (see Table 2), ranging from large tags (e.g. E. coli thioredoxin [12LaVallie E.R DiBlasio E.A Kovacic S Grant K.L Schendel P.F McCoy J.M A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm.BioTechnology. 1993; 11: 187-193Crossref PubMed Scopus (796) Google Scholar], Schistosoma japonicum glutathione-S-transferase [GST] [13Smith D.B Johnson K.S Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione-S-transferase.Gene. 1988; 67: 31-40Crossref PubMed Scopus (4986) Google Scholar] and E. coli maltose-binding protein [MBP] [14di Guan C Li P Riggs P.D Inouye H Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein.Gene. 1988; 67: 21-30Crossref PubMed Scopus (534) Google Scholar]) down to fairly small tags (e.g. S-tag [15Kim J-S Raines R.T Ribonuclease S-peptide as a carrier in fusion proteins.Protein Sci. 1993; 2: 348-356Crossref PubMed Scopus (176) Google Scholar], His-tag [16Hochuli E Bannwarth W Dobeli H Gentz R Stuber D Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent.BioTechnology. 1988; 6: 1321-1325Crossref Scopus (934) Google Scholar] and T7-tag) [17Uhlen M Moks T Gene fusions for purposes of expression: an introduction.Methods Enzymol. 1990; 185: 129-143Crossref PubMed Scopus (137) Google Scholar, 18Ford C.F Suominen I Glatz C.E Fusion tails for the recovery and purification of recombinant proteins.Protein Express. Purif. 1991; 2: 95-107Crossref PubMed Scopus (131) Google Scholar, 19Nilsson B Forsberg G Moks T Hartmanis M Uhlen M Fusion proteins in biotechnology and structural biology.Curr. Opin. Struct. Biol. 1992; 2: 569-575Crossref Scopus (44) Google Scholar, 20LaVallie E.R McCoy J.M Gene fusion expression systems in Escherichia coli.Curr. Opin. Biotechnol. 1995; 6: 501-506Crossref PubMed Scopus (186) Google Scholar, 21Nilsson J Stahl S Lundeberg J Uhlen M Nygren P-A Affinity fusion strategies for detection, purification, and immobilization of recombinant proteins.Protein Express. Purif. 1997; 11: 1-16Crossref PubMed Scopus (265) Google Scholar, 22Hannig G Makrides S.C Strategies for optimizing heterologous protein expression in Escherichia coli.Trends Biotechnol. 1998; 16: 54-60Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 23Berne P.F Doublie S Carter Jr., C.W Molecular biology for structural biology.in: Ducruix A Giege R Crystallization of Nucleic Acids and Proteins. A Practical Approach. Oxford University Press, Oxford1999: 45-73Google Scholar]. Although expression data and correlation to crystallization have not been conducted in a thorough manner, it is generally believed that the expression fusion tags need to be removed, particularly for large fusion partners. Table 3 summarizes the possible choices for incorporating tag-cleavage and self-cleavage into expression constructs. Unfortunately, the endopeptidases suffer from many limitations, including the presence of peptide secondary cleavage site activity (leading to proteolytically damaged products), incomplete sample cleavage (leading to product heterogeneity which hampers crystallization), and inhibition of cleavage by properly folded proteins (requiring partial denaturation for successful fusion-tail cleavage) [24LaVallie, E.R., McCoy, J.M., Smith, D.B. & Riggs, P. (1994). Enzymatic and chemical cleavage of fusion proteins. In Current Protocols in Molecular Biology. pp. 16.4.5–16.4.17, John Wiley and Sons, Inc, New York, NY.Google Scholar]. The viral proteases to date have proven to be the most selective and useful for structural biology studies. There is the additional possibility of utilizing expression constructs that incorporate different combinations of tags, for multiple affinity purification procedures that allow for increased selectivity of purification [8Makrides S.C Strategies for achieving high-level expression of genes in Escherichia coli.Microbiol. Rev. 1996; 60: 512-538Crossref PubMed Google Scholar, 17Uhlen M Moks T Gene fusions for purposes of expression: an introduction.Methods Enzymol. 1990; 185: 129-143Crossref PubMed Scopus (137) Google Scholar, 18Ford C.F Suominen I Glatz C.E Fusion tails for the recovery and purification of recombinant proteins.Protein Express. Purif. 1991; 2: 95-107Crossref PubMed Scopus (131) Google Scholar, 21Nilsson J Stahl S Lundeberg J Uhlen M Nygren P-A Affinity fusion strategies for detection, purification, and immobilization of recombinant proteins.Protein Express. Purif. 1997; 11: 1-16Crossref PubMed Scopus (265) Google Scholar, 25Kim J-S Raines R.T Peptide tags for a dual affinity fusion system.Anal. Biochem. 1994; 219: 165-166Crossref PubMed Scopus (25) Google Scholar, 26Müller K.M Arndt K.M Bauer K Plückthun A Tandem immobilized metal-ion affinity chromatography/immunoaffinity purification of His-tagged proteins — evaluation of two anti-His-tag monoclonal antibodies.Anal. Biochem. 1998; 259: 54-61Crossref PubMed Scopus (68) Google Scholar, 27Cocca B.A Seal S.N Radic M.Z Tandem affinity tags for the purification of bivalent anti-DNA single-chain Fv expressed in Escherichia coli.Protein Express. Purif. 1999; 17: 290-298Crossref PubMed Scopus (10) Google Scholar].Table 2Fusion tags used for recombinant protein expression and purification.TagSizeFusion tag locationTag typeCommentsHis-tag6, 8 or 10 aaN, C, internalPurificationMost common purification tag used for immobilized metal-affinity chromatography (IMAC) one-step purification 81Crowe J Döbeli H Gentz R Hochuli E Stüber D Henco K 6xHis-Ni-NTA chromatography as a superior technique in recombinant protein expression/purification.in: Harwood A.J Methods in Molecular Biology. Vol. 31. Humana Press Inc, Totawa1994: 371-387Google Scholar.Purification possible even under denaturing conditions 82Sherwood R Protein fusions: bioseparations and application.Trends Biotechnol. 1991; 9: 1-3Abstract Full Text PDF PubMed Scopus (13) Google Scholar.Tag possibly influences crystallization.T7-tag11 or 16 aaN, internalPurification, enhanced expressionMonoclonal antibody-based purification (denaturing low pH elution needed). Leaves unnatural N-terminal amino acids on the recombinant protein. Possibly enhanced expression levels as the T7-tag is derived from the T7 gene 10, which is the naturally most abundant phage T7 gene product.S-tag15 aaN, C, internalPurification and detectionS-protein (104 aa, ribonuclease A minus S-tag peptide sequence) modified resin affinity purification. RNAse S assay possible for quantitative assay of expression levels.FLAG™ peptide (DYKDDDDK)8 aaN, CPurificationCa2+-dependent monoclonal antibody purification with EDTA elution. Tag cleavable with enterokinase 83Hopp T.P Conlon P.J et al.A short polypeptide marker sequence useful for recombinant protein identification and purification.Bio/Technology. 1988; 6: 1204-1210Crossref Scopus (726) Google Scholar.Thioredoxin109 aa (11.7 kDa)N, CPurification and enhanced expressionAffinity purification with phenylarsine oxide-modified (ThioBond) resin.His-patch thioredoxin109 aa (11.7 kDa)N, CPurification and enhanced expressionUse of His-patch modified thioredoxin for IMAC purification 84Lu Z McCoy J.M et al.Histidine patch thioredoxins.J. Biol. Chem. 1996; 271: 5059-5065Crossref PubMed Scopus (65) Google Scholar.lacZ (β-galactosidase)116 kDaN, CPurificationPurification using p-amino-phenyl-β-d-thiogalactoside-modified sepharose. Classical tag used for protecting peptides from proteolytic degradation. Fusion proteins with this tag have a high tendency to be insoluble. Active enzyme is a tetramer.Chloramphenicol acetyltransferase24 kDaNSecretion, purification and detectionChloramphenicol–sepharose purification. Enzymatic assay possible for quantitation.trpE27 kDaNPurificationOften form insoluble precipitates. Hydrophobic interaction chromatography purification.Avidin/streptavidin/Strep-tagPurification and secretionBiotin affinity purification and streptavidin affinity purification (Strep-tag) 85Schmidt T.G.M Skerra A The random peptide library-assisted engineering of a C-terminal affinity peptide, useful for the detection and purification of a functional Ig Fv fragment.Protein Eng. 1993; 6: 109-122Crossref PubMed Scopus (249) Google Scholar.T7gene10260 aaNPurification and enhanced expressionProduces insoluble fusion protein (potential enhanced expression for toxic clones).Staphylococcal protein A14 kDa (or 31 kDa)NPurification and secretionIgG antibody affinity purification possible (denaturing low pH elution needed). Fusion protein secretion due to protein A signal sequence 86Nilsson B Abrahmsen L Uhlen M Immobilization and purification of enzymes with staphylococcal protein A gene fusion vectors.EMBO J. 1985; 4: 1075-1080Crossref PubMed Scopus (259) Google Scholar.Streptococcal protein G28 kDaN, CPurification and secretionAlbumin affinity purification, low pH elution needed. Fusion protein secretion due to protein G signal sequence.Glutathione-S-transferase (GST)26 kDaNPurificationGlutathione affinity or GST antibody purification. Enzymatic activity assay possible for quantitative analysis. Fusion proteins form dimers.Dihydrofolate reductase (DHFR)25 kDaNPurificationMethotrexate-linked agarose used for purification.Cellulose-binding domains (CBP)156 aa/NPurification and secretionCellulose-based resins used for affinity purification with water elution 87Greenwood J.M Gilkes N.R Kilburn D.G Miller Jr., R.C Warren R.A.J Fusion to an endoglucanase allows alkaline phosphatase to bind to cellulose.FEBS Lett. 1989; 244: 127-131Crossref PubMed Scopus (53) Google Scholar, 88Ong E Gilkes N.R Warren R.A.J Miller Jr., R.C Kilburn D.G Enzyme immobilization using the cellulose-binding domain of a Cellulomonas fimi exoglucanase.BioTechnol. 1989; 7: 604-607Crossref Scopus (74) Google Scholar. Different constructs available for cytoplasmic or periplasmic expression. Fusion proteins susceptible to proteolysis between the fusion partners 89Greenwood J.M Ong E Gilkes N.R Warren R.A.J Miller Jr., R.C Kilburn D.G Cellulose-binding domains: potential for purification of complex proteins.Protein Eng. 1992; 5: 361-365Crossref PubMed Scopus (39) Google Scholar.114 aa/N107 aa/CMaltose-binding protein (MBP)40 kDaN, CPurification and secretionAmylose affinity purification with maltose elution.Galactose-binding proteinPurificationGalactose-sepharose purification.Calmodulin-binding protein (CBP)4 kDaN, CPurification and detectionCalmodulin/Ca2+ affinity purification with EDTA elution.Can potentially assay expression levels with 32P-cAMP kinase.Hemagglutinin influenza virus (HAI)PurificationGreen fluorescent protein220 aaN, CDetectionUsed as reporter gene fusion for detection purposes 90Chalfie M Tu Y Euskirchen G Ward W.W Prasher D.C Green fluorescent protein as a marker for gene expression.Science. 1994; 263: 802-805Crossref PubMed Scopus (5286) Google Scholar.Used at one time for possible refolding tag.HSV-tag11 aaCPurificationMonoclonal antibody-based purification (denaturing low pH elution needed).B-tag (VP7 protein region of bluetongue virus)PurificationAnti-BTag antibody purification.Polyarginine5–15 aaCPurificationS-sepharose (cationic resin) purification. Fusion proteins potentially insoluble.Polycysteine4 aaNPurificationThiopropyl-sepharose purification.Polyphenylalanine11 aaNPurificationPhenyl-superose (hydrophobic interaction chromatography) purification.(Ala-Trp-Trp-Pro)nPurificationPolyaspartic acid5–16 aaCPurificationAnionic resin purification.KSI125 aaNEnhanced expressionHigh-level inclusion body production.c-mycPurificationAnti-myc antibody purification.OmpT/OmpA22 aa/21 aaNSecretionPeriplasmic leader sequences for potential protein export and folding 91Ghrayeb J Kimura H Takahara M Hsiung H Masui Y Inouye M Secretion cloning vectors in Escherichia coli.EMBO J. 1984; 3: 2437-2442Crossref PubMed Scopus (282) Google Scholar, as well as potential disulfide bond formation and isomerization./PelB/20 aa/DsbA/DsbC/208 aa/236 aaChitin-binding domainN, CExpressionUsed in the ImpactTM system, with intein-based expression constructs.NusA495 aaNPossible enhanced solubilityPotentially improve solubility for proteins that are overexpressed.Ubiquitin76 aaNPossible enhanced solubilityUbiquitin fusions observed to increase E. coli expressed recombinant protein solubility.lac repressorPurificationlac operator affinity purification.T4 gp55Growth hormone, N terminus Open table in a new tab Table 3Cleavage sites used in recombinant protein expression and purification.Excision site (↓)Cleavage enzyme/self-cleavageCommentsAsp-Asp-Asp-Asp-Lys↓EnterokinaseThe site will not cleave if followed by a proline residue.Secondary cleavage sites at other basic residues, depending on conformation of protein substrate. Active from pH4.5 to 9.5 and between 4°C and 45°C 24LaVallie, E.R., McCoy, J.M., Smith, D.B. & Riggs, P. (1994). Enzymatic and chemical cleavage of fusion proteins. In Current Protocols in Molecular Biology. pp. 16.4.5–16.4.17, John Wiley and Sons, Inc, New York, NY.Google Scholar.Ile-Glu/Asp-Gly-Arg↓Factor Xa proteaseWill not cleave if followed by proline and arginine.Secondary cleavage sites following Gly-Arg sequences.Leu-Val-Pro-Arg↓Gly−?SerThrombinSecondary cleavage sites. Biotinylated form available for removal with immobilized streptavidin.Glu-Asn-Leu-Tyr-Phe-Gln↓GlyTEV proteaseSeven-residue recognition site, making it a highly site-specific protease.Active over a wide range of temperatures.Protease available as a His-tag fusion protein, allowing for protease removal after recombinant protein cleavage.Leu-Glu-Val-Leu-Phe-Gln↓Gly-ProPreScission™ proteaseGenetically engineered form of human rhinovirus 3C protease with a GST fusion tag, allowing for facile cleavage and purification of GST-tagged proteins along with protease removal after recombinant protein cleavage.Enables low-temperature cleavage of fusion proteins containing the eight-residue recognition sequence.Specific intein-encoded sequencesIntein 1 and intein 2Uses self-cleavable affinity tags.Even after cleavage unnatural termini are present on the protein of interest.Signal sequencesSignal peptidasesCleavage of leader sequences concomitant with protein export from the cytoplasm. Open table in a new tab High-throughput expression requires the parallel induction of all clones in one expression run under the same conditions, which requires the presence of N-terminal expression tags to standardize baseline recombinant protein expression levels. In order to enable reliable prediction of expression constructs that generate soluble or insoluble expressed proteins, empirical trials need to be performed, altering expression conditions (e.g. the temperature or inducer concentration used for a run) and observing the solubilities and stabilities of the recombinant proteins, that are obtained [28Riggs, P., LaVallie, R. & McCoy, J.M. (1994). Introduction to expression by fusion protein vectors. In Current Protocols in Molecular Biology. pp. 16.4.1–16.4-4, John Wiley and Sons, Inc, New York, NY.Google Scholar]. As outlined in Figure 1, a probable combination of five different conditions should be probed. Prescreening using SDS–PAGE, in combination with Western blot analysis, is advantageous to analyze expression constructs on a small-scale and to determine the levels of proteins produced. This screening also provides information on the degradation or aggregation of proteins from potential expression clones. Host strain genotype is important for obtaining optimal expression levels, and many specialized E. coli strains have been developed. For example, the BL21 lon and ompT protease-deficient strain improves the likelihood of isolating intact full-length recombinant proteins [10Studier F.W Moffatt B.A Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes.J. Mol. Biol. 1986; 189: 113-130Crossref PubMed Scopus (4699) Google Scholar]. Another important variable is the induction level used [29Meerman H.J Georgiou G Construction and characterization of a set of E. coli strains deficient in all known loci affecting the proteolytic stability of secreted recombinant proteins.BioTechnology. 1994; 12: 1107-1110Crossref PubMed Scopus (119) Google Scholar]. Although high-level expression can usually be obtained, optimizing the yield of properly folded protein might require a reduction in the induction level. Finally, media formulations must be considered. In some instances, optimal expression might require supplementary salts (e.g. Zn2+, Cu2+, Ca2+, Fe2+, Fe3+) and potential cofactors or prosthetic groups (e.g. heme, FAD, FMN, tetrahydrobiopterin) to be included [30Mitraki A King J Protein folding intermediates and inclusion body formation.BioTechnol. 1989; 7: 690-697Crossref Scopus (424) Google Scholar]; for example, for proteins with catalytically or structurally important metal centers expression trials should contain metal ions in the culture media [31Goodwill K Sabatier C Stevens R.C The crystal structure of tyrosine hydroxylase with bound co-factor analog and iron at 2.3 Å resolution: the self-hydroxylation of Phe300 and the pterin binding site.Biochemistry. 1998; 37: 13437-13445Crossref PubMed Scopus (121) Google Scholar, 32Hanson M.A Stevens R.C Cocrystal structure of synaptobrevin-II bound to botulinum neurotoxin type B at 2.0 Å resolution.Nat. Struct. Biol. 2000; 7: 687-692Crossref PubMed Scopus (138) Google Scholar]. Studies should determine optimal media formulations for maximal recombinant protein yield; for example, with rich media formulations more active proteins are sometimes produced [33Moore J.T Arvinder U Maley F Maley G.F Overcoming inclusion body formation in a high level expression system.Protein Express. Purif. 1993; 4: 160-163Crossref PubMed Scopus (103) Google Scholar]. Current media conditions are old and outdated, and were developed for different applications. There is a need to develop new media conditions optimized for high-expression level protein production under high-density growth conditions. Recombinant protein purification is facilitated by the use of high-yield expression systems so that the desired protein is produced in an enriched form. Purification is further simplified by the presence of affinity purification tags. For high-throughput processing, initial efforts have focused on soluble and insoluble purification strategies using the cost-effective His-tag [34Arnold F.H Metal-affinity separations: a new dimension in protein processing.BioTechnology. 1991; 9: 151-156Crossref PubMed Scopus (462) Google Scholar]. The incorporation of a