Skip to main content
SpringerLink
Log in

A Brief Guide to the High-Throughput Expression of Directed Evolution Libraries

  • Protocol
  • First Online:
Protein Engineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1685))

  • 4886 Accesses

Abstract

The process of protein production optimization requires time and labor, constituting one of the main bottlenecks for the downstream utilization of the proteins. However, once through this bottleneck, the protein production process can be easily standardized and multiplexed to find the fittest variants in large libraries created by random mutagenesis. In this chapter, we present an overview of the most important choices to achieve homogeneous and functional expression of directed evolution libraries in microplate format: (1) choice of induction system and host strain, (2) choice of media and growth conditions, and (3) modifications to the genetic sequence.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Institutional subscriptions

References

  1. Graslund S, Nordlund P, Weigelt J et al (2008) Protein production and purification. Nat Methods 5:135–146

    Article  PubMed  Google Scholar 

  2. Demain AL, Vaishnav P (2009) Production of recombinant proteins by microbes and higher organisms. Biotechnol Adv 27:297–306

    Article  CAS  PubMed  Google Scholar 

  3. Makrides SC (1996) Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev 60:512–538

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Sørensen HP, Mortensen KK (2005) Advanced genetic strategies for recombinant protein expression in Escherichia coli. J Biotechnol 115:113–128

    Article  PubMed  Google Scholar 

  5. Costa S, Almeida A, Castro A et al (2014) Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: the novel Fh8 system. Front Microbiol 5:63

    PubMed  PubMed Central  Google Scholar 

  6. Studier FW, Rosenberg AH, Dunn JJ et al (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185:60–89

    Article  CAS  PubMed  Google Scholar 

  7. Dubendorff JW, Studier FW (1991) Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. J Mol Biol 219:45–59

    Article  CAS  PubMed  Google Scholar 

  8. Studier FW, Moffatt BA (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189:113–130

    Article  CAS  PubMed  Google Scholar 

  9. Zhang X, Studier FW (1997) Mechanism of inhibition of bacteriophage T7 RNA polymerase by T7 lysozyme. J Mol Biol 269:10–27

    Article  CAS  PubMed  Google Scholar 

  10. Wagner S, Klepsch MM, Schlegel S et al (2008) Tuning Escherichia coli for membrane protein overexpression. Proc Natl Acad Sci U S A 105:14371–14376

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Miroux B, Walker JE (1996) Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol 260:289–298

    Article  CAS  PubMed  Google Scholar 

  12. Novy R, Drott D, Yaeger K et al (2001) Overcoming the codon bias of E. coli for enhanced protein expression. Innov 12:1–3

    Google Scholar 

  13. Prinz WA, Aslund F, Holmgren A et al (1997) The role of the thioredoxin and glutaredoxin pathways in reducing protein disulfide bonds in the Escherichia coli cytoplasm. J Biol Chem 272:15661–15667

    Article  CAS  PubMed  Google Scholar 

  14. Ferrer M, Chernikova TN, Timmis KN, Golyshin PN (2004) Expression of a temperature-sensitive esterase in a novel chaperone-based Escherichia coli strain. Appl Environ Microbiol 70:4499–4504

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Baneyx F, Mujacic M (2004) Recombinant protein folding and misfolding in Escherichia coli. Nat Biotechnol 22:1399–1408

    Article  CAS  PubMed  Google Scholar 

  16. Studier FW (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41:207–234

    Article  CAS  PubMed  Google Scholar 

  17. Deutscher J (2008) The mechanisms of carbon catabolite repression in bacteria. Curr Opin Microbiol 11:87–93

    Article  CAS  PubMed  Google Scholar 

  18. Correa A, Oppezzo P (2011) Tuning different expression parameters to achieve soluble recombinant proteins in E. coli: advantages of high-throughput screening. Biotechnol J 6:715–730

    Article  CAS  PubMed  Google Scholar 

  19. Nilsson J, Stahl S, Lundeberg J et al (1997) Affinity fusion strategies for detection, purification, and immobilization of recombinant proteins. Protein Expr Purif 11:1–16

    Article  CAS  PubMed  Google Scholar 

  20. Wood DW (2014) New trends and affinity tag designs for recombinant protein purification. Curr Opin Struct Biol 26:54–61

    Article  CAS  PubMed  Google Scholar 

  21. Correa A, Oppezzo P (2015) Overcoming the solubility problem in E. coli: available approaches for recombinant protein production. Methods Mol Biol 1258:27–44

    Article  CAS  PubMed  Google Scholar 

  22. Bolanos-Garcia VM, Davies OR (2006) Structural analysis and classification of native proteins from E. coli commonly co-purified by immobilised metal affinity chromatography. Biochim Biophys Acta 1760:1304–1313

    Article  CAS  PubMed  Google Scholar 

  23. Schmidt TG, Skerra A (2007) The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins. Nat Protoc 2:1528–1535

    Article  CAS  PubMed  Google Scholar 

  24. Skerra A, Schmidt TG (2000) Use of the Strep-Tag and streptavidin for detection and purification of recombinant proteins. Methods Enzymol 326:271–304

    Article  CAS  PubMed  Google Scholar 

  25. Kapust RB, Waugh DS (1999) Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci 8:1668–1674

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Kellermann OK, Ferenci T (1982) Maltose-binding protein from Escherichia coli. Methods Enzymol 90:459–463

    Article  CAS  PubMed  Google Scholar 

  27. Nikaido H (1994) Maltose transport system of Escherichia coli: an ABC-type transporter. FEBS Lett 346:55–58

    Article  CAS  PubMed  Google Scholar 

  28. Bach H, Mazor Y, Shaky S et al (2001) Escherichia coli maltose-binding protein as a molecular chaperone for recombinant intracellular cytoplasmic single-chain antibodies. J Mol Biol 312:79–93

    Article  CAS  PubMed  Google Scholar 

  29. Smith DB, Johnson KS (1988) Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67:31–40

    Article  CAS  PubMed  Google Scholar 

  30. Kaplan W, Husler P, Klump H, Erhardt J, Sluis-Cremer N, Dirr H (1997) Conformational stability of pGEX-expressed Schistosoma japonicum glutathione S-transferase: a detoxification enzyme and fusion-protein affinity tag. Protein Sci 6:399–406

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Malhotra A (2009) Tagging for protein expression. Methods Enzymol 463:239–258

    Article  CAS  PubMed  Google Scholar 

  32. Hammarstrom M, Hellgren N, van Den Berg S et al (2002) Rapid screening for improved solubility of small human proteins produced as fusion proteins in Escherichia coli. Protein Sci 11:313–321

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Dyson MR, Shadbolt SP, Vincent KJ et al (2004) Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression. BMC Biotechnol 4:32

    Article  PubMed  PubMed Central  Google Scholar 

  34. Boyer TD (1989) The glutathione S-transferases: an update. Hepatology 9:486–496

    Article  CAS  PubMed  Google Scholar 

  35. LaVallie ER, DiBlasio EA, Kovacic S et al (1993) A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Nat Biotechnol 11:187–193

    Article  CAS  Google Scholar 

  36. LaVallie ER, Lu Z, Diblasio-Smith EA et al (2000) Thioredoxin as a fusion partner for production of soluble recombinant proteins in Escherichia coli. Methods Enzymol 326:322–340

    Article  CAS  PubMed  Google Scholar 

  37. Kim S, Lee SB (2008) Soluble expression of archaeal proteins in Escherichia coli by using fusion-partners. Protein Expr Purif 62:116–119

    Article  CAS  PubMed  Google Scholar 

  38. Terpe K (2003) Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 60:523–533

    Article  CAS  PubMed  Google Scholar 

  39. Dummler A, Lawrence AM, de Marco A (2005) Simplified screening for the detection of soluble fusion constructs expressed in E. coli using a modular set of vectors. Microb Cell Fact 4:34

    Article  PubMed  PubMed Central  Google Scholar 

  40. Gusarov I, Nudler E (2001) Control of intrinsic transcription termination by N and NusA: the basic mechanisms. Cell 107:437–449

    Article  CAS  PubMed  Google Scholar 

  41. Marblestone JG, Edavettal SC, Lim Y et al (2006) Comparison of SUMO fusion technology with traditional gene fusion systems: enhanced expression and solubility with SUMO. Protein Sci 15:182–189

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. De Marco V, Stier G, Blandin S et al (2004) The solubility and stability of recombinant proteins are increased by their fusion to NusA. Biochem Biophys Res Commun 322:766–771

    Article  PubMed  Google Scholar 

  43. Davis GD, Elisee C, Newham DM et al (1999) New fusion protein systems designed to give soluble expression in Escherichia coli. Biotechnol Bioeng 65:382–388

    Article  CAS  PubMed  Google Scholar 

  44. Butt TR, Edavettal SC, Hall JP (2005) SUMO fusion technology for difficult-to-express proteins. Protein Expr Purif 43:1–9

    Article  CAS  PubMed  Google Scholar 

  45. Li SJ, Hochstrasser M (1999) A new protease required for cell-cycle progression in yeast. Nature 398:246–251

    Article  CAS  PubMed  Google Scholar 

  46. Khorasanizadeh S, Peters ID, Roder H (1996) Evidence for a three-state model of protein folding from kinetic analysis of ubiquitin variants with altered core residues. Nat Struct Biol 3:193–205

    Article  CAS  PubMed  Google Scholar 

  47. Malakhov MP, Mattern MR, Malakhova OA et al (2004) SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins. J Struct Funct Genomics 5:75–86

    Article  CAS  PubMed  Google Scholar 

  48. Nallamsetty S, Waugh DS (2007) Mutations that alter the equilibrium between open and closed conformations of Escherichia coli maltose-binding protein impede its ability to enhance the solubility of passenger proteins. Biochem Biophys Res Commun 364:639–644

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Douette P, Navet R, Gerkens P et al (2005) Escherichia coli fusion carrier proteins act as solubilizing agents for recombinant uncoupling protein 1 through interactions with GroEL. Biochem Biophys Res Commun 333:686–693

    Article  CAS  PubMed  Google Scholar 

  50. Fox JD, Kapust RB, Waugh DS (2001) Single amino acid substitutions on the surface of Escherichia coli maltose-binding protein can have a profound impact on the solubility of fusion proteins. Protein Sci 10:622–630

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Zhang YB, Howitt J, McCorkle S et al (2004) Protein aggregation during overexpression limited by peptide extensions with large net negative charge. Protein Expr Purif 36:207–216

    Article  CAS  PubMed  Google Scholar 

  52. Su Y, Zou Z, Feng S et al (2007) The acidity of protein fusion partners predominantly determines the efficacy to improve the solubility of the target proteins expressed in Escherichia coli. J Biotechnol 129:373–382

    Article  CAS  PubMed  Google Scholar 

  53. Esposito D, Chatterjee DK (2006) Enhancement of soluble protein expression through the use of fusion tags. Curr Opin Biotechnol 17:353–358

    Article  CAS  PubMed  Google Scholar 

  54. Arnau J, Lauritzen C, Petersen GE, Pedersen J (2006) Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins. Protein Expr Purif 48:1–13

    Article  CAS  PubMed  Google Scholar 

  55. Pina AS, Lowe CR, Roque AC (2014) Challenges and opportunities in the purification of recombinant tagged proteins. Biotechnol Adv 32:366–381

    Article  CAS  PubMed  Google Scholar 

  56. Koehn J, Hunt I (2009) High-throughput protein production (HTPP): a review of enabling technologies to expedite protein production. Methods Mol Biol 498:1–18

    Article  CAS  PubMed  Google Scholar 

  57. Young CL, Britton ZT, Robinson AS (2012) Recombinant protein expression and purification: a comprehensive review of affinity tags and microbial applications. Biotechnol J 7:620–634

    Article  CAS  PubMed  Google Scholar 

  58. Waugh DS (2011) An overview of enzymatic reagents for the removal of affinity tags. Protein Expr Purif 80:283–293

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Blommel PG, Becker KJ, Duvnjak P et al (2007) Enhanced bacterial protein expression during auto-induction obtained by alteration of lac repressor dosage and medium composition. Biotechnol Prog 23:585–598

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Kensy F, Engelbrecht C, Büchs J (2009) Scale-up from microtiter plate to laboratory fermenter: evaluation by online monitoring techniques of growth and protein expression in Escherichia coli and Hansenula polymorpha fermentations. Microb Cell Fact 8:68

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Molecular Biology, Center for Molecular Biology “Severo Ochoa” (UAM-CSIC), Universidad Autónoma de Madrid, Nicolas Cabrera 1, Madrid, 28049, Spain

    Ana Luísa Ribeiro, Mario Mencía & Aurelio Hidalgo

Authors
  1. Ana Luísa Ribeiro
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mario Mencía
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Aurelio Hidalgo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Aurelio Hidalgo .

Editor information

Editors and Affiliations

  1. Department of Biotechnology and Enzyme Catalysis Institute of Biochemistry, Greifswald University, Greifswald, Germany

    Uwe T. Bornscheuer

  2. Protein Biochemistry Institute of Biochemistry, Greifswald University, Greifswald, Germany

    Matthias Höhne

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media LLC

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Ribeiro, A.L., Mencía, M., Hidalgo, A. (2018). A Brief Guide to the High-Throughput Expression of Directed Evolution Libraries. In: Bornscheuer, U., Höhne, M. (eds) Protein Engineering. Methods in Molecular Biology, vol 1685. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7366-8_7

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7366-8_7

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7364-4

  • Online ISBN: 978-1-4939-7366-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation