Screening Fusion Tags for Improved Recombinant Protein Expression in E. coli with the Expresso® Solubility and Expression Screening System

Eric J. Steinmetz1, Michele E. Auldridge1

1 Lucigen Corporation, Middleton, Wisconsin
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 5.27
DOI:  10.1002/cpps.39
Online Posting Date:  November, 2017
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Abstract

The simplicity, speed, and low cost of bacterial culture make E. coli the system of choice for most initial trials of recombinant protein expression. However, many heterologous proteins are either poorly expressed in bacteria, or are produced as incorrectly folded, insoluble aggregates that lack the activity of the native protein. In many cases, fusion to a partner protein can allow for improved expression and/or solubility of a difficult target protein. Although several different fusion partners have gained favor, none are universally effective, and identifying the one that best improves soluble expression of a given target protein is an empirical process. This unit presents a strategy for parallel screening of fusion partners for enhanced expression or solubility. The Expresso® Solubility and Expression Screening System includes a panel of seven distinct fusion partners and utilizes an extremely simple cloning strategy to enable rapid screening and identification of the most effective fusion partner. © 2017 by John Wiley & Sons, Inc.

Keywords: autoinduction; expresso; fusion tag; protein expression; solubility

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Cloning a Target Gene into pSol Vectors
  • Support Protocol 1: Screening Clones by Colony PCR
  • Basic Protocol 2: Testing Expression and Solubility of Fusion Proteins
  • Alternate Protocol 1: Autoinduction
  • Basic Protocol 3: Purification of Fusion Proteins by Metal Affinity Chromatography
  • Basic Protocol 4: Removal of Solubility Tag by Cleavage with SelecTEV Protease
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Cloning a Target Gene into pSol Vectors

  Materials
  • Custom primers for amplification of gene encoding protein of interest
  • Expresso® Solubility and Expression Screening System kit containing:
  • Pre‐processed pSol vectors
    • E. cloni 10G chemically competent cells
    • Recovery Medium
    • DNA encoding protein of interest
  • Proofreading PCR polymerase and reaction buffer
  • LB‐Lennox plates containing 30 µg/ml kanamycin (see recipe)
  • Ice
  • 15‐ml disposable polypropylene culture tubes (17 × 100 mm)
  • 42°C water bath
  • 37°C shaking incubator
  • 37°C plate incubator
  • Additional reagents and equipment for PCR (see appendix 4J; Kramer & Coen, ) and agarose gel electrophoresis (see appendix 4F; Voytas, )

Support Protocol 1: Screening Clones by Colony PCR

  Materials
  • Expresso® Solubility and Expression Screening System kit containing:
    • pRham Forward primer
    • pETite Reverse primer
  • CloneID 1× Colony PCR Master Mix (e.g., Lucigen)
  • Plates containing candidate colonies (see protocol 1)
  • Molecular weight markers
  • PCR strip tubes
  • Programmable thermal cycler
  • Additional reagents and equipment for agarose gel electrophoresis (see appendix 4F; Voytas, )

Basic Protocol 2: Testing Expression and Solubility of Fusion Proteins

  Materials
  • Colonies with verified clones (see protocol 1 and protocol 2)
  • LB‐Miller liquid medium containing 30 μg/ml kanamycin (see recipe)
  • 2× SDS‐PAGE sample loading buffer (see recipe)
  • Expresso® Solubility and Expression Screening System kit containing:
    • 20% rhamnose solution
  • Cell lysis buffer (see recipe)
  • 10× SDS‐PAGE gel running buffer (see recipe)
  • 15‐well 4% to 20% polyacrylamide gel (e.g., Bio‐Rad MiniProtean TGX)
  • Molecular weight markers
  • Coomassie brilliant blue R250 gel staining solution (see recipe)
  • 37°C shaking incubator
  • Spectrophotometer and disposable spectrophotometer cuvettes
  • Centrifuge (e.g., Sorvall RC‐5)
  • Microcentrifuge
  • 1.5‐ml microcentrifuge tubes
  • 95°C heating block
  • Sonicator equipped with a 1/8 in. micro‐tip or with an 8‐tip sonicator horn (e.g., Qsonica, cat. no. 4602)
  • Additional reagents and equipment for SDS‐PAGE (see unit 10.1; Gallagher, )

Alternate Protocol 1: Autoinduction

  Materials
  • Expresso® Solubility and Expression Screening System kit containing:
    • 20% rhamnose solution
    • 15% glucose solution
  • LB‐Miller liquid medium containing 30 μg/ml kanamycin (see recipe)
  • Culture or colony of interest (see protocol 1 and protocol 2)
  • 37°C shaking incubator
  • Spectrophotometer

Basic Protocol 3: Purification of Fusion Proteins by Metal Affinity Chromatography

  Materials
  • Cleared cell lysate from induced culture (see protocol 3)
  • Ni‐NTA Resin (e.g., Qiagen)
  • Cell lysis buffer (see recipe)
  • Column wash buffer (see recipe)
  • Elution buffer (see recipe)
  • 2× SDS‐PAGE sample loading buffer (see recipe)
  • 4% to 20% polyacrylamide gel (e.g., Bio‐Rad MiniProtean TGX)
  • Coomassie brilliant blue R250 gel staining solution (see recipe)
  • Dialysis buffer (see recipe)
  • Bradford Protein Assay reagents (e.g., Thermo Fisher Scientific)
  • Sonicator
  • Centrifuge
  • Chromatography column (e.g., Bio‐Rad, cat. no. 731‐1550)
  • 95°C heating block
  • Dialysis tubing
  • Spectrophotometer
  • Additional reagents and equipment for SDS‐PAGE (see unit 10.1; Gallagher, ) and protein quantification (see Simonian & Smith, )

Basic Protocol 4: Removal of Solubility Tag by Cleavage with SelecTEV Protease

  Materials
  • Purified fusion protein (see protocol 5)
  • Expresso® Solubility and Expression Screening System kit containing (or available separately; e.g., Lucigen):
    • 20× SelecTEV protease buffer
    • 100 mM DTT
    • 10 U/µl SelecTEV protease
  • 2× SDS‐PAGE sample loading buffer (see recipe)
  • 4% to 20% polyacrylamide gel (e.g., Bio‐Rad MiniProtean TGX)
  • Coomassie brilliant blue R250 gel staining solution (see recipe)
  • Ni‐NTA Resin (e.g., Qiagen)
  • 95°C heating block
  • Chromatography column (e.g., Bio‐Rad, cat. no. 731‐1550)
  • Additional reagents and equipment for SDS‐PAGE (see unit 10.1; Gallagher, )
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Figures

Videos

Literature Cited

  Bubeck, P., Winkler, M., & Bautsch, W. (1993). Rapid cloning by homologous recombination in vivo. Nucleic Acids Research, 21, 3601–3602. doi: 10.1093/nar/21.15.3601.
  Butt, T. R., Jonnalagadda, S., Monia, B. P., Sternberg, E. J., Marsh, J. A., Stadel, J. M., & Crooke, S. T. (1989). Ubiquitin fusion augments the yield of cloned gene products in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America, 86, 2540–2544. doi: 10.1073/pnas.86.8.2540.
  Egan, S. M., & Schleif, R. F. (1993). A regulatory cascade in the induction of rhaBAD. Journal of Molecular Biology, 234, 87–98. doi: 10.1006/jmbi.1993.1565.
  Esposito, D., & Chatterjee, D. K. (2006). Enhancement of soluble protein expression through the use of fusion tags. Current Opinion in Biotechnology, 17, 353–358. doi: 10.1016/j.copbio.2006.06.003.
  Gallagher, S. R. 2012. One‐dimensional SDS gel electrophoresis of proteins. Current Protocols in Protein Science, 68, 10.1.1–10.1.44. doi: 10.1002/0471140864.ps1001s68.
  Giacalone, M. J., Gentile, A. M., Lovitt, B. T., Berkley, N. L., Gunderson, C. W., & Surber, M. W. (2006). Toxic protein expression in Escherichia coli using a rhamnose‐based tightly regulated and tunable promoter system. Biotechniques, 40, 355–364. doi: 10.2144/000112112.
  Gibson, D. G., Young, L., Chuang, R. Y., Venter, J. C., Hutchison, C. A. 3rd, & Smith, H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 6, 343–345. doi: 10.1038/nmeth.1318.
  Goulet, A., Spinelli, S., Blangy, S., van Tilbeurgh, H., Leulliot, N., Basta, T., … Campanacci, V. (2009). The thermo‐ and acido‐stable ORF‐99 from the archaeal virus AFV1. Protein Science, 18, 1316–1320. doi: 10.1002/pro.122.
  Haldimann, A., Daniels, L. L., & Wanner, B. L. (1998). Use of new methods for construction of tightly regulated arabinose and rhamnose promoter fusions in studies of the Escherichia coli phosphate regulon. Journal of Bacteriology, 180, 1277–1286.
  Han, K. Y., Song, J. A., Ahn, K. Y., Park, J. S., Seo, H. S., & Lee, J. (2007a). Enhanced solubility of heterologous proteins by fusion expression using stress‐induced Escherichia coli protein, Tsf. FEMS Microbiology Letters, 274, 132–138. doi: 10.1111/j.1574‐6968.2007.00824.x.
  Han, K. Y., Song, J. A., Ahn, K. Y., Park, J. S., Seo, H. S., & Lee, J. (2007b). Solubilization of aggregation‐prone heterologous proteins by covalent fusion of stress‐responsive Escherichia coli protein, SlyD. Protein Engineering, Design & Selection, 20, 543–549. doi: 10.1093/protein/gzm055.
  Kapust, R. B., & Waugh, D. S. (1999). Escherichia coli maltose‐binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Science, 8, 1668–1674. doi: 10.1110/ps.8.8.1668.
  Kapust, R. B., Tözsér, J., Fox, J. D., Anderson, D. E., Cherry, S., Copeland, T. D., & Waugh, D. S. (2001). Tobacco etch virus protease: Mechanism of autolysis and rational design of stable mutants with wild‐type catalytic proficiency. Protein Engineering, 14, 993–1000. doi: 10.1093/protein/14.12.993.
  Kramer, M. F. & Coen, D. M. (2002). The polymerase chain reaction. Current Protocols in Protein Science, 29, A.4J.1–A.4J.8. doi: 10.1002/0471140864.psa04js29.
  LaVallie, E. R., DiBlasio, E. A., Kovacic, S., Grant, K. L., Schendel, P. F., & McCoy, J. M. (1993). A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Biotechnology, 11, 187–193. doi: 10.1038/nbt0293‐187.
  Marblestone, J. G., Edavettal, S. C., Lim, Y., Lim, P., Zuo, X., & Butt, T. R. (2006). Comparison of SUMO fusion technology with traditional gene fusion systems: Enhanced expression and solubility with SUMO. Protein Science, 15, 182–189. doi: 10.1110/ps.051812706.
  Nallamsetty, S., Kapust, R. B., Tozser, J., Cherry, S., Tropea, J. E., Copeland, T. D., & Waugh, D. S. (2004). Efficient site‐specific processing of fusion proteins by tobacco vein mottling virus protease in vivo and in vitro. Protein Expression and Purification, 38, 108–115. doi: 10.1016/j.pep.2004.08.016.
  Parks, T. D., Leuther, K. K., Howard, E. D., Johnston, S. A., & Dougherty, W. G. (1994). Release of proteins and peptides from fusion proteins using a recombinant plant virus proteinase. Analytical Biochemistry, 216, 413–417. doi: 10.1006/abio.1994.1060.
  Phan, J., Zdanov, A., Evdokimov, A. G., Tropea, J. E., Peters, H. K., 3rd, Kapust, R. B., & Waugh, D. S. (2002). Structural basis for the substrate specificity of tobacco etch virus protease. Journal of Biological Chemistry, 277, 50564–50572. doi: 10.1074/jbc.M207224200.
  Pryor, K. D., & Leiting, B. (1997). High‐level expression of soluble protein in Escherichia coli using a His6‐tag and maltose‐binding‐protein double‐affinity fusion system. Protein Expression and Purification, 10, 309–319. doi: 10.1006/prep.1997.0759.
  Raran‐Kurussi, S., & Waugh, D. S. (2012). The ability to enhance the solubility of its fusion partners is an intrinsic property of maltose‐binding protein but their folding is either spontaneous or chaperone‐mediated. PLoS One, 7, e49589. doi: 10.1371/journal.pone.0049589.
  Simonian, M. H. & Smith, J. A. (2006). Spectrophotometric and colorimetric determination of protein concentration. Current Protocols in Molecular Biology, 76, 10.1A.1–10.1A.9. doi: 10.1002/0471142727.mb1001as76.
  Smith, D. B., & Johnson, K. S. (1988). Single‐step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S‐transferase. Gene, 67, 31–40. doi: 10.1016/0378‐1119(88)90005‐4.
  Tokunaga, H., Saito, S., Sakai, K., Yamaguchi, R., Katsuyama, I., Arakawa, T., & Tokunaga, M. (2010). Halophilic beta‐lactamase as a new solubility‐ and folding‐enhancing tag protein: Production of native human interleukin 1alpha and human neutrophil alpha‐defensin. Applied Microbiology and Biotechnology, 86, 649–658. doi: 10.1007/s00253‐009‐2325‐9.
  Voytas, D. (2001). Agarose gel electrophoresis. Current Protocols in Protein Science, 13, A.4F.1–A.4F.3. doi: 10.1002/0471140864.psa04fs13.
  Wegerer, A., Sun, T., & Altenbuchner, J. (2008). Optimization of an E. coli L‐rhamnose‐inducible expression vector: Test of various genetic module combinations. BMC Biotechnology, 8, 2. doi: 10.1186/1472‐6750‐8‐2.
  Zhang, Y. B., Howitt, J., McCorkle, S., Lawrence, P., Springer, K., & Freimuth, P. (2004). Protein aggregation during overexpression limited by peptide extensions with large net negative charge. Protein Expression and Purification, 36, 207–216. doi: 10.1016/j.pep.2004.04.020.
  Zou, Z., Cao, L., Zhou, P., Su, Y., Sun, Y., & Li, W. (2008). Hyper‐acidic protein fusion partners improve solubility and assist correct folding of recombinant proteins expressed in Escherichia coli. Journal of Biotechnology, 135, 333–339. doi: 10.1016/j.jbiotec.2008.05.007.
Internet Resources
  https://www.lucigen.com/Protein‐Expression/
  Additional background on the Expresso® Solubility and Expression Screening System and related Expresso® products, including vector sequence information.
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