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Codon Optimizing for Increased Membrane Protein Production: A Minimalist Approach

  • Kiavash Mirzadeh
  • Stephen Toddo
  • Morten H. H. Nørholm
  • Daniel O. Daley
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1432)

Abstract

Reengineering a gene with synonymous codons is a popular approach for increasing production levels of recombinant proteins. Here we present a minimalist alternative to this method, which samples synonymous codons only at the second and third positions rather than the entire coding sequence. As demonstrated with two membrane-embedded transporters in Escherichia coli, the method was more effective than optimizing the entire coding sequence. The method we present is PCR based and requires three simple steps: (1) the design of two PCR primers, one of which is degenerate; (2) the amplification of a mini-library by PCR; and (3) screening for high-expressing clones.

Key words

Membrane protein Protein expression Codon optimization Synonymous codon 

Notes

Acknowledgments

This work was supported by a grant from the Swedish Research Council to DOD, and by the Novo Nordisk Foundation to MHHN.

Conflict of interest statement: The method considered here has been described in a patent submitted by CloneOpt AB (SE1451553-0). KM, ST, and DOD are founders and shareholders in CloneOpt AB.

References

  1. 1.
    Gustafsson C, Minshull J, Govindarajan S, Ness J, Villalobos A, Welch M (2012) Engineering genes for predictable protein expression. Protein Expr Purif 83:37–46CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Bulmer M (1987) Coevolution of codon usage and transfer RNA abundance. Nature 325:728–730CrossRefPubMedGoogle Scholar
  3. 3.
    Ikemura T (1981) Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes: a proposal for a synonymous codon choice that is optimal for the E. coli translational system. J Mol Biol 151:389–409CrossRefPubMedGoogle Scholar
  4. 4.
    Norholm MH, Light S, Virkki MT, Elofsson A, von Heijne G, Daley DO (2012) Manipulating the genetic code for membrane protein production: what have we learnt so far? Biochim Biophys Acta 1818:1091–1096CrossRefPubMedGoogle Scholar
  5. 5.
    Norholm MH, Toddo S, Virkki MT, Light S, von Heijne G, Daley DO (2013) Improved production of membrane proteins in Escherichia coli by selective codon substitutions. FEBS Lett 587:2352–2358CrossRefPubMedGoogle Scholar
  6. 6.
    Stenstrom CM, Jin H, Major LL, Tate WP, Isaksson LA (2001) Codon bias at the 3'-side of the initiation codon is correlated with translation initiation efficiency in Escherichia coli. Gene 263:273–284CrossRefPubMedGoogle Scholar
  7. 7.
    Mirzadeh K, Martinez V, Toddo S, Guntur S, Herrgard MJ, Elofsson A, Norholm MH, Daley DO (2015) Enhanced protein production in Escherichia coli by optimization of cloning scars at the vector-coding sequence junction. ACS Synth Biol 4(9):959–965CrossRefPubMedGoogle Scholar
  8. 8.
    Laursen BS, Sorensen HP, Mortensen KK, Sperling-Petersen HU (2005) Initiation of protein synthesis in bacteria. Microbiol Mol Biol Rev 69:101–123CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    McCarthy JE, Gualerzi C (1990) Translational control of prokaryotic gene expression. Trends Genet 6:78–85CrossRefPubMedGoogle Scholar
  10. 10.
    Kudla G, Murray AW, Tollervey D, Plotkin JB (2009) Coding-sequence determinants of gene expression in Escherichia coli. Science 324:255–258CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Plotkin JB, Kudla G (2011) Synonymous but not the same: the causes and consequences of codon bias. Nat Rev Genet 12:32–42CrossRefPubMedGoogle Scholar
  12. 12.
    Mortimer SA, Kidwell MA, Doudna JA (2014) Insights into RNA structure and function from genome-wide studies. Nat Rev Genet 15:469–479CrossRefPubMedGoogle Scholar
  13. 13.
    Ding Y, Tang Y, Kwok CK, Zhang Y, Bevilacqua PC, Assmann SM (2014) In vivo genome-wide profiling of RNA secondary structure reveals novel regulatory features. Nature 505:696–700CrossRefPubMedGoogle Scholar
  14. 14.
    Daley DO, Rapp M, Granseth E, Melen K, Drew D, von Heijne G (2005) Global topology analysis of the Escherichia coli inner membrane proteome. Science 308:1321–1323CrossRefPubMedGoogle Scholar
  15. 15.
    Drew D, Lerch M, Kunji E, Slotboom DJ, de Gier JW (2006) Optimization of membrane protein overexpression and purification using GFP fusions. Nat Methods 3:303–313CrossRefPubMedGoogle Scholar
  16. 16.
    Kimchi-Sarfaty C, Oh JM, Kim IW, Sauna ZE, Calcagno AM, Ambudkar SV, Gottesman MM (2007) A "silent" polymorphism in the MDR1 gene changes substrate specificity. Science 315:525–528CrossRefPubMedGoogle Scholar
  17. 17.
    Kim SJ, Yoon JS, Shishido H, Yang Z, Rooney LA, Barral JM, Skach WR (2015) Protein folding. Translational tuning optimizes nascent protein folding in cells. Science 348:444–448CrossRefPubMedGoogle Scholar
  18. 18.
    Ellis HM, Yu D, DiTizio T, Court DL (2001) High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. PNAS 98:6742–6746CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Sorensen HP, Mortensen KK (2005) Advanced genetic strategies for recombinant protein expression in Escherichia coli. J Biotechnol 115:113–128CrossRefPubMedGoogle Scholar
  20. 20.
    Rosano GL, Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 5:172PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Kiavash Mirzadeh
    • 1
  • Stephen Toddo
    • 1
  • Morten H. H. Nørholm
    • 2
  • Daniel O. Daley
    • 1
  1. 1.Department of Biochemistry and Biophysics, Center for Biomembrane ResearchStockholm UniversityStockholmSweden
  2. 2.Novo Nordisk Foundation Center for BiosustainabilityTechnical University of DenmarkLyngbyDenmark

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