Skip to main content
SpringerLink
Log in

Rational-Based Protein Engineering: Tips and Tools

  • Protocol
  • First Online:
Protein Nanotechnology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 996))

Abstract

The rational engineering of proteins is driven by contemporary needs for new and altered biomolecular forms. Utilizing manipulative procedures of molecular biology, it is relatively straightforward to alter protein structure and function to create mutated or fused sequences. We here give an overview of procedures and strategies for site-directed mutagenesis, construction of fusion proteins, and insertion of tags. The design of new protein constructs as well as their over-expression as recombinant products is considered. We also summarize approaches for the engineering of protein complexes by co-expression, a valuable route to generate bioactive multicomponent systems.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Grunberg R, Serrano L (2010) Strategies for protein synthetic biology. Nucleic Acids Res 38:2663–2675

    Article  PubMed  Google Scholar 

  2. Graslund S, Sagemark J, Berglund H, Dahlgren LG, Flores A, Hammarstrom M, Johansson I, Kotenyova T, Nilsson M, Nordlund P, Weigelt J (2008) The use of systematic N- and C-terminal deletions to promote production and structural studies of recombinant proteins. Protein Expr Purif 58:210–221

    Article  PubMed  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  4. Woestenenk EA, Hammarstrom M, van den Berg S, Hard T, Berglund H (2004) His-tag effect on solubility of human proteins produced in Escherichia coli: a comparison between four expression vectors. J Struct Funct Genomics 5:217–229

    Article  PubMed  CAS  Google Scholar 

  5. Gavin AC, Superti-Furga G (2003) Protein complexes and proteome organization from yeast to man. Curr Opin Chem Biol 7:21–27

    Article  PubMed  CAS  Google Scholar 

  6. Ahnert SE, Teichmann SA (2008) Networks for all. Genome Biol 9:324

    Article  PubMed  Google Scholar 

  7. Tolia NH, Joshua-Tor L (2006) Strategies for protein co-expression in Escherichia coli. Nat Methods 3:55–64

    Article  PubMed  CAS  Google Scholar 

  8. Romier C, Ben Jelloul M, Albeck S, Buchwald G, Busso D, Celie PH, Christodoulou E, De Marco V, van Gerwen S, Knipscheer P, Lebbink JH, Notenboom V, Poterszman A, Rochel N, Cohen SX, Unger T, Sussman JL, Moras D, Sixma TK, Perrakis A (2006) Co-expression of protein complexes in prokaryotic and eukaryotic hosts: experimental procedures, database tracking and case studies. Acta Crystallogr D Biol Crystallogr 62:1232–1242

    Article  PubMed  Google Scholar 

  9. Perrakis A, Romier C (2008) Assembly of protein complexes by co-expression in prokaryotic and eukaryotic hosts: an overview. Methods Mol Biol 426:247–256

    Article  PubMed  CAS  Google Scholar 

  10. Palomares LA, Estrada-Mondaca S, Ramírez OT (2004) Production of Recombinant Proteins: challenges and solutions. Methods Mol Biol 267:15–52

    PubMed  CAS  Google Scholar 

  11. Aricescu AR, Assenberg R, Bill RM, Busso D, Chang VT, Davis SJ, Dubrovsky A, Gustafsson L, Hedfalk K, Heinemann U, Jones IM, Ksiazek D, Lang C, Maskos K, Messerschmidt A, Macieira S, Peleg Y, Perrakis A, Poterszman A, Schneider G, Sixma TK, Sussman JL, Sutton G, Tarboureich N, Zeev-Ben-Mordehai T, Jones EY (2006) Eukaryotic expression: developments for structural proteomics. Acta Crystallogr D Biol Crystallogr 62:1114–1124

    Article  PubMed  CAS  Google Scholar 

  12. Endo Y, Sawasaki T (2006) Cell-free expression systems for eukaryotic protein production. Curr Opin Biotechnol 17:373–380

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  14. Sivashanmugam A, Murray V, Cui C, Zhang Y, Wang J, Li Q (2009) Practical protocols for production of very high yields of recombinant proteins using Escherichia coli. Protein Sci 18:936–948

    Article  PubMed  CAS  Google Scholar 

  15. Welch M, Villalobos A, Gustafsson C, Minshull J (2009) You’re one in a googol: optimizing genes for protein expression. J R Soc Interface 6(Suppl 4):S467–S476

    Article  PubMed  CAS  Google Scholar 

  16. Kane JF (1995) Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli. Curr Opin Biotechnol 6:494–500

    Article  PubMed  CAS  Google Scholar 

  17. Chen GF, Inouye M (1990) Suppression of the negative effect of minor arginine codons on gene expression; preferential usage of minor codons within the first 25 codons of the Escherichia coli genes. Nucleic Acids Res 18:1465–1473

    Article  PubMed  CAS  Google Scholar 

  18. Fuhrmann M, Hausherr A, Ferbitz L, Schodl T, Heitzer M, Hegemann P (2004) Monitoring dynamic expression of nuclear genes in Chlamydomonas reinhardtii by using a synthetic luciferase reporter gene. Plant Mol Biol 55:869–881

    PubMed  CAS  Google Scholar 

  19. Sharp PM, Li WH (1987) The codon Adaptation Index–a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Res 15:1281–1295

    Article  PubMed  CAS  Google Scholar 

  20. Kane JF, Violand BN, Curran DF, Staten NR, Duffin KL, Bogosian G (1992) Novel in-frame two codon translational hop during synthesis of bovine placental lactogen in a recombinant strain of Escherichia coli. Nucleic Acids Res 20:6707–6712

    Article  PubMed  CAS  Google Scholar 

  21. Puigbo P, Guzman E, Romeu A, Garcia-Vallve S (2007) OPTIMIZER: a web server for optimizing the codon usage of DNA sequences. Nucleic Acids Res 35:126–131

    Article  Google Scholar 

  22. Dieci G, Bottarelli L, Ballabeni A, Ottonello S (2000) tRNA-assisted overproduction of eukaryotic ribosomal proteins. Protein Expr Purif 18:346–354

    Article  PubMed  CAS  Google Scholar 

  23. Yuan L, Kurek I, English J, Keenan R (2005) Laboratory-directed protein evolution. Microbiol Mol Biol Rev 69:373–392

    Article  PubMed  CAS  Google Scholar 

  24. Shimada A (1996) PCR-Based Site-Directed Mutagenesis Vol. 57, Methods Mol Med 57: 157–165

    Google Scholar 

  25. Xu Z, Colosimo A, Gruenert DC (2003) Site-directed mutagenesis using the megaprimer method. Methods Mol Biol 235:203–207

    PubMed  CAS  Google Scholar 

  26. Dominy CN, Andrews DW (2003) Site-directed mutagenesis by inverse PCR. Methods Mol Biol 235:209–223

    PubMed  CAS  Google Scholar 

  27. Lee J, Shin MK, Ryu DK, Kim S, Ryu WS (2010) Insertion and deletion mutagenesis by overlap extension PCR. Methods Mol Biol 634:137–146

    Article  PubMed  CAS  Google Scholar 

  28. Sambrook J (2000) Molecular cloning: A laboratory manual. CSH press, America

    Google Scholar 

  29. Lewis MK, Thompson DV (1990) Efficient site directed in vitro mutagenesis using ampicillin selection. Nucleic Acids Res 18:3439–3443

    Article  PubMed  CAS  Google Scholar 

  30. Deng WP, Nickoloff JA (1992) Site-directed mutagenesis of virtually any plasmid by eliminating a unique site. Anal Biochem 200:81–88

    Article  PubMed  CAS  Google Scholar 

  31. Kunkel TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA 82:488–492

    Article  PubMed  CAS  Google Scholar 

  32. Taylor JW, Ott J, Eckstein F (1985) The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res 13:8765–8785

    Article  PubMed  CAS  Google Scholar 

  33. Klock HE, Koesema EJ, Knuth MW, Lesley SA (2008) Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts. Proteins 71:982–994

    Article  PubMed  CAS  Google Scholar 

  34. Graslund S, Nordlund P, Weigelt J, Hallberg BM, Bray J, Gileadi O, Knapp S, Oppermann U, Arrowsmith C, Hui R, Ming J, dhe-Paganon S, Park HW, Savchenko A, Yee A, Edwards A, Vincentelli R, Cambillau C, Kim R, Kim SH, Rao Z, Shi Y, Terwilliger TC, Kim CY, Hung LW, Waldo GS, Peleg Y, Albeck S, Unger T, Dym O, Prilusky J, Sussman JL, Stevens RC, Lesley SA, Wilson IA, Joachimiak A, Collart F, Dementieva I, Donnelly MI, Eschenfeldt WH, Kim Y, Stols L, Wu R, Zhou M, Burley SK, Emtage JS, Sauder JM, Thompson D, Bain K, Luz J, Gheyi T, Zhang F, Atwell S, Almo SC, Bonanno JB, Fiser A, Swaminathan S, Studier FW, Chance MR, Sali A, Acton TB, Xiao R, Zhao L, Ma LC, Hunt JF, Tong L, Cunningham K, Inouye M, Anderson S, Janjua H, Shastry R, Ho CK, Wang D, Wang H, Jiang M, Montelione GT, Stuart DI, Owens RJ, Daenke S, Schutz A, Heinemann U, Yokoyama S, Bussow K, Gunsalus KC (2008) Protein production and purification. Nat Methods 5:135–146

    Article  PubMed  Google Scholar 

  35. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    Article  PubMed  CAS  Google Scholar 

  36. Finn RD, Tate J, Mistry J, Coggill PC, Sammut SJ, Hotz HR, Ceric G, Forslund K, Eddy SR, Sonnhammer EL, Bateman A (2008) The Pfam protein families database. Nucleic Acids Res 36:D281–D288

    Article  PubMed  CAS  Google Scholar 

  37. Apweiler R, Bairoch A, Wu CH, Barker WC, Boeckmann B, Ferro S, Gasteiger E, Huang H, Lopez R, Magrane M, Martin MJ, Natale DA, O’Donovan C, Redaschi N, Yeh LS (2004) UniProt: the Universal Protein knowledgebase. Nucleic Acids Res 32:D115–D119

    Article  PubMed  CAS  Google Scholar 

  38. Kanehisa M, Goto S, Kawashima S, Nakaya A (2002) The KEGG databases at GenomeNet. Nucleic Acids Res 30:42–46

    Article  PubMed  CAS  Google Scholar 

  39. Edgar RC, Batzoglou S (2006) Multiple sequence alignment. Curr Opin Struct Biol 16:368–373

    Article  PubMed  CAS  Google Scholar 

  40. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE (2000) The Protein Data Bank. Nucleic Acids Res 28:235–242

    Article  PubMed  CAS  Google Scholar 

  41. Kiefer F, Arnold K, Kunzli M, Bordoli L, Schwede T (2009) The SWISS-MODEL Repository and associated resources. Nucleic Acids Res 37:D387–D392

    Article  PubMed  CAS  Google Scholar 

  42. Linding R, Russell RB, Neduva V, Gibson TJ (2003) GlobPlot: Exploring protein sequences for globularity and disorder. Nucleic Acids Res 31:3701–3708

    Article  PubMed  CAS  Google Scholar 

  43. Gao X, Bain K, Bonanno JB, Buchanan M, Henderson D, Lorime D, Marsh C, Reynes JA, Sauder JM, Schwinn K, Thai C, Burley SK (2005) High-throughput limited proteolysis/mass spectrometry for protein domain elucidation. J Struct Funct Genomics 6:129–134

    Article  PubMed  CAS  Google Scholar 

  44. Hamuro Y, Coales SJ, Southern MR, Nemeth-Cawley JF, Stranz DD, Griffin PR (2003) Rapid analysis of protein structure and dynamics by hydrogen/deuterium exchange mass spectrometry. J Biomol Tech 14:171–182

    PubMed  Google Scholar 

  45. Farr GW, Furtak K, Rowland MB, Ranson NA, Saibil HR, Kirchhausen T, Horwich AL (2000) Multivalent binding of nonnative substrate proteins by the chaperonin GroEL. Cell 100:561–573

    Article  PubMed  CAS  Google Scholar 

  46. Robinson CR, Sauer RT (1998) Optimizing the stability of single-chain proteins by linker length and composition mutagenesis. Proc Natl Acad Sci USA 95:5929–5934

    Article  PubMed  CAS  Google Scholar 

  47. Volkel T, Korn T, Bach M, Muller R, Kontermann RE (2001) Optimized linker sequences for the expression of monomeric and dimeric bispecific single-chain diabodies. Protein Eng 14:815–823

    Article  PubMed  CAS  Google Scholar 

  48. Sobti M, Cubeddu L, Haynes PA, Mabbutt BC (2010) Engineered rings of mixed yeast Lsm proteins show differential interactions with translation factors and U-rich RNA. Biochemistry 49:2335–2345

    Article  PubMed  CAS  Google Scholar 

  49. Wurch T, Lestienne F, Pauwels PJ (1998) A modified overlap extension PCR method to create chimeric genes in the absence of restriction enzymes. Biotechnol Tech 12:653–657

    Article  CAS  Google Scholar 

  50. Hearn MT, Acosta D (2001) Applications of novel affinity cassette methods: use of peptide fusion handles for the purification of recombinant proteins. J Mol Recognit 14:323–369

    Article  PubMed  CAS  Google Scholar 

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

    PubMed  CAS  Google Scholar 

  52. Uhlen M, Nilsson B, Guss B, Lindberg M, Gatenbeck S, Philipson L (1983) Gene fusion vectors based on the gene for staphylococcal protein A. Gene 23:369–378

    Article  PubMed  CAS  Google Scholar 

  53. Walls D, Loughran ST (2011) Tagging recombinant proteins to enhance solubility and aid purification. Methods Mol Biol 681:151–175

    Article  PubMed  CAS  Google Scholar 

  54. Rais-Beghdadi C, Roggero MA, Fasel N, Reymond CD (1998) Purification of recombinant proteins by chemical removal of the affinity tag. Appl Biochem Biotechnol 74:95–103

    Article  PubMed  CAS  Google Scholar 

  55. Walker PA, Leong LE, Ng PW, Tan SH, Waller S, Murphy D, Porter AG (1994) Efficient and rapid affinity purification of proteins using recombinant fusion proteases. Biotechnology (N Y) 12:601–605

    Article  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  57. Drew DE, von Heijne G, Nordlund P, de Gier JW (2001) Green fluorescent protein as an indicator to monitor membrane protein overexpression in Escherichia coli. FEBS Lett 507:220–224

    Article  PubMed  CAS  Google Scholar 

  58. Korepanova A, Moore JD, Nguyen HB, Hua Y, Cross TA, Gao F (2007) Expression of membrane proteins from Mycobacterium tuberculosis in Escherichia coli as fusions with maltose binding protein. Protein Expr Purif 53:24–30

    Article  PubMed  CAS  Google Scholar 

  59. Roosild TP, Greenwald J, Vega M, Castronovo S, Riek R, Choe S (2005) NMR structure of Mistic, a membrane-integrating protein for membrane protein expression. Science 307:1317–1321

    Article  PubMed  CAS  Google Scholar 

  60. Loughran ST, Walls D (2011) Purification of Poly-Histidine-Tagged Proteins. Methods Mol Biol 681:311–335

    Article  PubMed  CAS  Google Scholar 

  61. Nilsson J, Larsson M, Stahl S, Nygren PA, Uhlen M (1996) Multiple affinity domains for the detection, purification and immobilization of recombinant proteins. J Mol Recognit 9:585–594

    Article  PubMed  CAS  Google Scholar 

  62. Prinz B, Schultchen J, Rydzewski R, Holz C, Boettner M, Stahl U, Lang C (2004) Establishing a versatile fermentation and purification procedure for human proteins expressed in the yeasts Saccharomyces cerevisiae and Pichia pastoris for structural genomics. J Struct Funct Genomics 5:29–44

    Article  PubMed  CAS  Google Scholar 

  63. Gingras AC, Gstaiger M, Raught B, Aebersold R (2007) Analysis of protein complexes using mass spectrometry. Nat Rev Mol Cell Biol 8:645–654

    Article  PubMed  CAS  Google Scholar 

  64. 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  PubMed  CAS  Google Scholar 

  65. Charlton A, Zachariou M (2011) Tag removal by site-specific cleavage of recombinant fusion proteins. Methods Mol Biol 681:349–367

    Article  PubMed  CAS  Google Scholar 

  66. Chong S, Mersha FB, Comb DG, Scott ME, Landry D, Vence LM, Perler FB, Benner J, Kucera RB, Hirvonen CA, Pelletier JJ, Paulus H, Xu MQ (1997) Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element. Gene 192:271–281

    Article  PubMed  CAS  Google Scholar 

  67. Wang HM, Shih YP, Hu SM, Lo WT, Lin HM, Ding SS, Liao HC, Liang PH (2009) Parallel gene cloning and protein production in multiple expression systems. Biotechnol Prog 25:1582–1586

    PubMed  CAS  Google Scholar 

  68. Eschenfeldt WH, Lucy S, Millard CS, Joachimiak A, Mark ID (2009) A family of LIC vectors for high-throughput cloning and purification of proteins. Methods Mol Biol 498:105–115

    Article  PubMed  CAS  Google Scholar 

  69. Sievert V, Ergin A, Bussow K (2008) High throughput cloning with restriction enzymes. Methods Mol Biol 426:163–173

    Article  PubMed  CAS  Google Scholar 

  70. Aslanidis C, de Jong PJ (1990) Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res 18:6069–6074

    Article  PubMed  CAS  Google Scholar 

  71. Abdullah JM, Joachimiak A, Collart FR (2009) “System 48” high-throughput cloning and protein expression analysis. Methods Mol Biol 498:117–127

    Article  PubMed  CAS  Google Scholar 

  72. Widersten M (1998) Heterologous expression in Escherichia coli of soluble active-site random mutants of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 by co-expression of molecular chaperonins GroEL/ES. Protein Expr Purif 13:389–395

    Article  PubMed  CAS  Google Scholar 

  73. Wakagi T, Oshima T, Imamura H, Matsuzawa H (1998) Cloning of the gene for inorganic pyrophosphatase from a thermoacidophilic archaeon, Sulfolobus sp. strain 7, and overproduction of the enzyme by co-expression of tRNA for arginine rare codon. Biosci Biotechnol Biochem 62:2408–2414

    Article  PubMed  CAS  Google Scholar 

  74. Kerrigan JJ, Xie Q, Ames RS, Lu Q (2011) Production of protein complexes via co-expression. Protein Expr Purif 75:1–14

    Article  PubMed  CAS  Google Scholar 

  75. Fribourg S, Romier C, Werten S, Gangloff YG, Poterszman A, Moras D (2001) Dissecting the interaction network of multi-protein complexes by pairwise co-expression of subunits in E. coli. J Mol Biol 306:363–373

    Article  PubMed  CAS  Google Scholar 

  76. Johnston K, Clements A, Venkataramani RN, Trievel RC, Marmorstein R (2000) Co-expression of proteins in bacteria using T7-based expression plasmids: expression of heteromeric cell-cycle and transcriptional regulatory complexes. Protein Expr Purif 20:435–443

    Article  PubMed  CAS  Google Scholar 

  77. Held D, Yaeger K, Novy R (2003) New co-expression vectors for expanded compatibilities in E. coli. Innovations 18:4–6

    Google Scholar 

  78. Novy R, Yaeger K, Held D, Mierendorf R (2002) Co-expression of multiple target proteins in E. coli. Innovations 15:2–6

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Structural and Computational Biology Division, Victor Chang Cardiac Research Institute, Sydney, Australia

    Meghna Sobti

  2. Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, Australia

    Bridget C. Mabbutt

Authors
  1. Meghna Sobti
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bridget C. Mabbutt
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. University of Canterbury, Kirkwood Avenue 20, Christchurch, 8041, New Zealand

    Juliet A. Gerrard

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media, New York

About this protocol

Cite this protocol

Sobti, M., Mabbutt, B.C. (2013). Rational-Based Protein Engineering: Tips and Tools. In: Gerrard, J. (eds) Protein Nanotechnology. Methods in Molecular Biology, vol 996. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-354-1_14

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-354-1_14

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-353-4

  • Online ISBN: 978-1-62703-354-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation