Scalable High-Throughput Micro-Expression Device for Recombinant Proteins
2004; Future Science Ltd; Volume: 37; Issue: 3 Linguagem: Inglês
10.2144/04373bm05
ISSN1940-9818
AutoresRebecca Page, Kin Moy, Eric Sims, Jeffrey Velasquez, Brendan McManus, Carina Grittini, Thomas Clayton, Raymond C. Stevens,
Tópico(s)Advanced Fluorescence Microscopy Techniques
ResumoBioTechniquesVol. 37, No. 3 BenchmarksOpen AccessScalable high-throughput micro-expression device for recombinant proteinsRebecca Page, Kin Moy, Eric C. Sims, Jeffrey Velasquez, Brendan McManus, Carina Grittini, Thomas L. Clayton & Raymond C. StevensRebecca PageJoint Center for Structural Genomics and Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA, USA, Kin MoyJoint Center for Structural Genomics and Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA, USA, Eric C. SimsJoint Center for Structural Genomics and Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA, USA, Jeffrey VelasquezJoint Center for Structural Genomics and Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA, USA, Brendan McManusJoint Center for Structural Genomics and Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA, USA, Carina GrittiniJoint Center for Structural Genomics and Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA, USA, Thomas L. ClaytonJoint Center for Structural Genomics and Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA, USA & Raymond C. Stevens*Address correspondence to: Raymond C. Stevens, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. e-mail: E-mail Address: stevens@scripps.eduJoint Center for Structural Genomics and Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA, USAPublished Online:6 Jun 2018https://doi.org/10.2144/04373BM05AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail The large-scale expression and purification of recombinant proteins needed for structural studies is time-consuming and expensive, especially when costly reagents, such as selenomethionine (SeMet) or 15N/13C-labeled nuclear magnetic resonance (NMR) medium, are required. Small-scale screens are being developed to identify which targets express and are soluble prior to large-scale expression (1–7). Although these screens are designed for a 96-sample, small-scale format, none adequately predicts the reliable expression behavior with scaled-up milliliter and liter fermentations, which has been a challenge for researchers in the field.We have adapted a low-cost, high-velocity incubating Glas-Col Vertiga shaker (Glas-Col, LLC, Terre Haute, IN, USA; Figure 1A) to develop an efficient, high-throughput Escherichia coli microliter-scale expression screening protocol that accurately predicts protein behavior expressed in large-scale (milliliter and liter) fermentation conditions. The apparatus shakes cultures in three dimensions at speeds of up to 1000 rpm, allowing small-scale (approximately 500 µL) cultures grown in 2-mL 96-deep-well blocks to achieve absorbances (A600) as high as 10–20. This generates sufficient material for the analysis of expression, solubility, and binding to affinity purification matrices. Moreover, this screening strategy has also been used to identify clones that express and are soluble under SeMet (8) or 15N/13C-labeled expression conditions that are necessary for the production of labeled recombinant proteins for direct structural analysis. The Glas-Col Vertiga shaker can be used to screen for soluble expression clones under native, SeMet, and 15N/13C-labeled expression conditions.Figure 1. Glas-Col Vertiga microliter and Genomics Institute of the Novartis Research Foundation (GNF) milliliter expression systems.(A) Glas-Col Vertiga microliter-scale expression system. A low-cost vertical shaker apparatus adapted for the development of a scalable high-throughput Escherichia coli micro-expression screening. (B) GNF 96-well milliliter fermenter (9). A typical run includes the growth of approximately 65 mL of growth media, grown to an absorbance (A600) of 10–20 using media conditions similar to the micro-liter-scale expression experiments, with oxygen supplementation.Microscale expression using the Glas-Col Vertiga shaker is carried out as follows. First, overnight cultures are prepared with 250 µL sterile 2xyt media containing antibiotic (0.3 mM ampicillin) in sterile 2-mL, 96-deep-well, round-bottom blocks (USA Scientific, Ocala, FL, USA). Each well is then inoculated with 1 µL glycerol stocks of expression clones (also stored in a 96-well format) using a multichannel pipet. Once sealed with a sterile, porous plate cover (USA Scientific), the blocks are placed in the Glas-Col Vertiga shaker, and cultures are grown overnight at 37°C, shaking at 550 rpm. Expression under native conditions is carried out using 500 µL sterile Terrific Broth (TB or 2× TB) media prepared with 100 mM MOPS, pH 7.4, and antibiotic, while expression under SeMet conditions is carried out using sterile complete media with 1 µg/mL FeSO4, 0.8 mM SeMet, and antibiotic. Blocks containing the overnight cultures are then centrifuged at 5000× g to pellet the E. coli cells, the supernatants are discarded, and the pellets are resus-pended in 100 µL fresh media (TB or SeMet, as appropriate). The freshly prepared expression blocks are then inoculated with 25 µL of the resuspended overnight culture. After sealing each expression block with a porous plate cover, blocks are placed in the GlasCol Vertiga shaker and grown at 900 rpm and 37°C. When the culture A600 reaches approximately 3 (about 1.5 h following inoculation at 37°C; spectrophotometer wavelength, 600 nm), expression is induced [final concentration of 0.2% (w/v) L-arabinose (Sigma, St. Louis, MO, USA)] and cultures are allowed to shake at 900 rpm and 37°C for an additional 4–5 h. The final average culture A600 is usually between 10 and 20. Blocks are then centrifuged at 5000× g for 10 min to pellet the cells, and the supernatants are discarded. Blocks with pelleted cells are then analyzed for expression or sealed with a rubber plate seal and stored at −80°C for future analysis. In addition to the L-arabinose expression system (pBAD vector; Invitrogen, Carlsbad, CA, USA), similar microliter-scale and milliliter/liter experiments have also been carried out with the T7 expression system (pET vectors; Novagen, Madison, WI, USA).Expression and solubility are evaluated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis (His-tag antibodies). First, frozen bacterial cell pellets are placed at room temperature and allowed to thaw slightly. Cells are then thoroughly resuspended in 100 µL of MP lysis buffer A [50 mM Tris, pH 7.5, 50 mM sucrose, 1 mM EDTA, 0.25 mM tris-(2-carboxyethyl) phosphine hydrochloride (TCEP)] with 1.0 mg/mL lysozyme and 0.25 µL/mL Benzonase® (Novagen). After incubating at room temperature for 15 min, 100 µL of MP lysis buffer B (10 mM Tris, pH 7.5, 50 mM KCl, 10 mM MgCl2, 1 mM EDTA, 0.25 mM TCEP) are added to each well and incubated for an additional 15 min. After mixing, 10 µL samples of the lysate from each well are removed and added to the SDS-PAGE sample buffer. These samples represent the "total lysate" fractions. Samples are then centrifuged at 6100× g for 15 min to pellet cell debris. Following centrifugation, 20 µL of the supernatant are removed for SDS-PAGE analysis. These samples represent the "soluble" fractions.If desired, the soluble fraction is then purified using immobilized metal-affinity chromatography (IMAC). A 400-µL, 0.45-µm filter-containing 96-well microplate is used for the load, wash, and elution steps, and a 450-µL 96-well plate is placed underneath the filter plate to collect the flow through. One hundred microliters of a 50% slurry of Co2+-charged metal-affinity resin (TALON™ Superflow; BD Biosciences Clontech, Palo Alto, CA, USA) are added to each well of the filter plate and equilibrated with 300 µL of equilibration buffer (1:1 mixture of MP lysis buffers A and B). Equilibration buffer is removed by centrifugation at 300× g for 1 min. Clarified lysate supernatants are then added to each well and gently mixed by slowly pipetting up and down for 10 repetitions. The resin is allowed to settle to the bottom of each well and is then centrifuged as before, with new collection plates underneath to collect soluble lysate flow-through fractions. The resin is then washed once with MP wash buffer (25 mM Tris, pH 7.8, 150 mM NaCl, 5 mM imidazole, 0.25 mM TCEP) and centrifuged as before, saving the flow-through fractions in a third plate. The bound proteins are eluted from the IMAC resin using MP elution buffer (25 mM Tris, pH 7.8, 150 mM NaCl, 150 mM imidazole, 0.25 mM TCEP). The resin is slowly resuspended with 50 µL of elution buffer, after which the resin beads are allowed to settle for 10 min. The filter plates are then centrifuged a final time, using a new 450-µL 96-well plate to collect the eluate. Purified proteins can be used for additional analytical studies, such as mass spectrometry or size exclusion chromatography, as desired. A complete analysis of recombinant protein expression is performed by running 4%–20% SDS-PAGE gels of the total lysate, soluble, flow-through (unbound), and eluate fractions.For comparison to larger-scale fermentation behavior, 34 proteins were expressed using the microliter-scale expression protocol under native conditions and expressed again using the Genomics Institute of the Novartis Research Foundation (GNF; San Diego, CA, USA) 96-well milliliter fermenter (Figure 1B). This experiment was carried out in duplicate. Targets expressed in the GNF 96-well fermenter were purified as outlined by Lesley, et al. (9), with purity and yield determined by SDS-PAGE. Most samples were further analyzed using Western blot analysis with an anti-His antibody (Sigma) using standard protocols. Of the 34 proteins tested, 32 consistently expressed (or did not express) in both microliter and larger-scale volumes, illustrating a high level of scalability and correlation in the expression levels of soluble protein between the Vertiga screening trials and large-scale growth conditions (Table 1). In contrast, without the Vertiga shaker, the correlation between microliter- and milliliter- (and liter) scale expression is much lower (data not shown). The two proteins that behaved differently between micro- and macro-expression, BH0832 (GenBank® accession no. 10173447) and AGR_L_2357p (accession no. 15159665), did so in only one of the two experiments; in the second, the micro- and macro-expression behavior was identical for both proteins (Table 1). Finally, eukaryotic proteins from Saccharomyces cerevisiae and viral proteins from the Severe Acute Respiratory Syndrome (SARS) virus have also been successfully micro-expressed in E. coli using this device.Table 1. Comparison List of Microliter-Expressed and Scaled-Up Expressed ProteinsThese results demonstrate that the microliter-scale expression protocol developed using the Vertiga vertical shaker can be used to accurately identify proteins that will express solubly in larger-scale fermentation conditions. Moreover, the results from these screens can be used to assess the solubility and expected protein yield for each potential protein target for both native and labeled E. coli recombinant expression. Future developments include using material directly from microliter-scale expression for biophysical analysis, including nanovolume crystallization (10) and other biophysical techniques currently being miniaturized.AcknowledgmentsThis work was supported in part by grant no. GM62411 (to I.A. Wilson, P.I., JCSG) from the National Institutes of Health (NIH) Protein Structure Initiative. We appreciate the assistance of Lee Clark, Jim Jasco from Glas-Col, Mark Knuth and Ciaran Cronin from Syrrx for the initial studies of the Vertiga system, and Scott Lesley, Mark Weselak, and Bob Downs for the GNF fermenter design.Competing Interests StatementThe authors declare that they have no competing interests.References1. Holz, C., O. Hesse, N. Bolotina, U. Stahl, and C. Lang. 2002. A micro-scale process for high-throughput expression of cDNAs in the yeast Saccharomyces cerevisiae. Protein Expr. Purif. 25:372–378.Crossref, Medline, CAS, Google Scholar2. Shih, Y.-P., W.-M. Kung, J.-C. Chen, C.-H. Yeh, A.H.-J. Wang, and T.-F. Wang. 2002. High-throughput screening of soluble recombinant proteins. Protein Sci. 11:1714–1719.Crossref, Medline, CAS, Google Scholar3. Adams, M.W.W., H.A. Dailey, L.J. DeLucas, M. Luo, J.H. Prestegard, J.P. Rose, and B.-C. Wang. 2003. The Southeast Collaboratory for Structural Genomics: a high-throughput gene to structure factory. Acc. Chem. Res. 36:191–198.Crossref, Medline, CAS, Google Scholar4. Nguyen, H., B. Martinez, N. Oganesyan, and K. Rosalind. 2004. An automated small-scale protein expression and purification screening provides beneficial information for protein production. J. Struct. Funct. Genomics 5:23–27.Crossref, Medline, CAS, Google Scholar5. Scheich, C., V. Sievert, and K. Büssow. 2003. An automated method for high-throughput protein purification applied to a comparison of His-tag and GST-tag affinity chromatography. BMC Biotechnol. 3:12–19.Crossref, Medline, Google Scholar6. Knaust, R.K.C. and P. Nordlund. 2001. Screening for soluble expression of recombinant proteins in a 96-well format. Anal. Biochem. 297:79–85.Crossref, Medline, CAS, Google Scholar7. Yokoyama, S. 2003. Protein expression systems for structural genomics and proteomics. Curr. Opin. Chem. Biol. 7:39–43.Crossref, Medline, CAS, Google Scholar8. Hendrickson, W.A., J.R. Horton, and D.M. LeMaster. 1990. Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three-dimensional structure. EMBO J. 9:1665–1672.Crossref, Medline, CAS, Google Scholar9. Lesley, S.A., P. Kuhn, A. Godzik, A.M. Deacon, I. Mathews, A. Kreusch, G. Spraggon, H.E. Klock, et al.. 2002. Structural genomics of the Thermotoga maritima proteome implemented in a high-throughput structure determination pipeline. Proc. Natl. Acad. Sci. USA 99:11664–11669.Crossref, Medline, CAS, Google Scholar10. Santarsiero, B.D., D.T. Yegian, C.C. Lee, G. Spraggon, J. Gu, D. Scheibe, D.C. Uber, E.W. Cornell, et al.. 2002. An approach to rapid protein crystallization using nanodroplets. J. Appl. Crystallogr. 35:278–281.Crossref, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByCloning, expression and purification of kinase domains of cacao PR-1 receptor-like kinasesProtein Expression and Purification, Vol. 146Structural Proteomics19 April 2018A Simple Method for Optimization of Reference Gene Identification and Normalization in DNA Microarray Analysis1 January 2016 | Medical Science Monitor Basic Research, Vol. 22Protein production from the structural genomics perspective: achievements and future needsCurrent Opinion in Structural Biology, Vol. 23, No. 3Parallel production and verification of protein products using a novel high-throughput screening method16 June 2011 | Biotechnology Journal, Vol. 6, No. 8A secretory system for bacterial production of high-profile protein targets24 February 2011 | Protein Science, Vol. 20, No. 3High-throughput expression and purification of membrane proteinsJournal of Structural Biology, Vol. 172, No. 1High-throughput production of human proteins for crystallization: The SGC experienceJournal of Structural Biology, Vol. 172, No. 1Codon optimization can improve expression of human genes in Escherichia coli: A multi-gene studyProtein Expression and Purification, Vol. 59, No. 1The use of systematic N- and C-terminal deletions to promote production and structural studies of recombinant proteinsProtein Expression and Purification, Vol. 58, No. 2Protein production and purification30 January 2008 | Nature Methods, Vol. 5, No. 2High Throughput Production of Recombinant Human Proteins for CrystallographyStrategies for Improving Crystallization Success RatesProtein Crystallization in Restricted Geometry: Advancing Old Ideas for Modern Times in Structural ProteomicsMicrocoil NMR Spectroscopy: a Novel Tool for Biological High Throughput NMR SpectroscopySmall-scale, semi-automated purification of eukaryotic proteins for structure determination6 November 2007 | Journal of Structural and Functional Genomics, Vol. 8, No. 4Profiling of membrane protein variants in a baculovirus system by coupling cell-surface detection with small-scale parallel expressionProtein Expression and Purification, Vol. 56, No. 1Biophysical characterization of vaccinia virus thymidine kinase substrate utilizationJournal of Virological Methods, Vol. 142, No. 1-2Remedial strategies in structural proteomics: Expression, purification, and crystallization of the Vav1/Rac1 complexProtein Expression and Purification, Vol. 53, No. 1Economical parallel protein expression screening and scale-up in Escherichia coli23 December 2006 | Journal of Structural and Functional Genomics, Vol. 7, No. 2Five Years of Increasing Structural Biology Throughput - A Retrospective AnalysisProtein CrystallizationStrategies to maximize heterologous protein expression in Escherichia coli with minimal costProtein Expression and Purification, Vol. 51, No. 1Multiple Microfermentor Battery: a Versatile Tool for Use with Automated Parallel Cultures of Microorganisms Producing Recombinant Proteins and for Optimization of Cultivation ProtocolsApplied and Environmental Microbiology, Vol. 72, No. 8Towards miniaturization of a structural genomics pipeline using micro-expression and microcoil NMR9 November 2005 | Journal of Structural and Functional Genomics, Vol. 6, No. 4Structural Genomics of the Severe Acute Respiratory Syndrome Coronavirus: Nuclear Magnetic Resonance Structure of the Protein nsP7Journal of Virology, Vol. 79, No. 20Baculovirus as versatile vectors for protein expression in insect and mammalian cells5 May 2005 | Nature Biotechnology, Vol. 23, No. 5 Vol. 37, No. 3 Follow us on social media for the latest updates Metrics Downloaded 651 times History Received 19 April 2004 Accepted 1 June 2004 Published online 6 June 2018 Published in print September 2004 Information© 2004 Author(s)AcknowledgmentsThis work was supported in part by grant no. GM62411 (to I.A. Wilson, P.I., JCSG) from the National Institutes of Health (NIH) Protein Structure Initiative. We appreciate the assistance of Lee Clark, Jim Jasco from Glas-Col, Mark Knuth and Ciaran Cronin from Syrrx for the initial studies of the Vertiga system, and Scott Lesley, Mark Weselak, and Bob Downs for the GNF fermenter design.Competing Interests StatementThe authors declare that they have no competing interests.PDF download
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