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Molecular Diagnosis of Infectious Diseases pp 257–292Cite as

Expression and Purification of Recombinant Proteins Using the pET System

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Part of the book series: Methods in Molecular Medicine™ ((MIMM,volume 13))

Abstract

The pET System is the most powerful system yet developed for the cloning and expression of recombinant proteins in Escherichia coli Target genes are cloned in pET plasmids under control of strong bacteriophage T7 transcription and (optionally) translation signals, expression is induced by providing a source of T7 RNA polymerase in the host cell (1–2). T7 RNA polymerase is so selective and active that almost all of the cell’s resources are converted to target gene expression; the desired product can comprise more than 50% of the total cell protein after a few hours of induction. Another important benefit of this system is its ability to maintain target genes transcriptionally silent in the uninduced state. Target genes are initially cloned using hosts that do not contain the T7 RNA polymerase gene, thus eliminating plasmid instability caused by the production of proteins potentially toxic to the host cell. Once established in a nonexpression host, plasmids are then transferred into expression hosts containing a chromosomal copy of the T7 RNA polymerase gene under lacUV5 control, and expression is induced by the addition of IPTG. Two types of T7 promoter and several hosts that differ in then- stringency of suppressing basal expression levels are available, providing great flexibility and optimizing the expression of a wide variety of target genes. This chapter describes the vectors, hosts, and basic protocols for cloning, expression, and purification of target proteins in the pET System

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References

  1. Studier F W and Moffatt B A. (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes J Mol Biol 189, 113–130

    Article  PubMed  CAS  Google Scholar 

  2. Rosenberg A H, Lade B N, Chui D, Lin S, Dunn J J, and Studier F. W (1987) Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene 56, 125–135

    Article  PubMed  CAS  Google Scholar 

  3. Studier F W, Rosenberg A H, Dunn J J, and Dubendorff J W (1990) Use of T7 RNA polymerase to direct the expression of cloned genes Meth Enzymol 185, 60–89

    Article  PubMed  CAS  Google Scholar 

  4. Aslanidis C. and de Jong P J (1990) Ligation-independent cloning of PCR products (LIC-PCR) Nucleic Acids Res 18, 6069–6074

    Article  PubMed  CAS  Google Scholar 

  5. Seed B. (1987) An LFA-3 cDNA encodes a phosphohpid-linked membrane protein homologous to its receptor CD2 Nature 329, 840–842

    Article  PubMed  CAS  Google Scholar 

  6. Dubendorff J W and Studier F W (1991) Creation of a T7 autogene Cloning and expression of the gene for bacteriophage T7 RNA polymerase under control of its cognate promoter J Mol Biol 219, 45–59

    Article  PubMed  CAS  Google Scholar 

  7. Grodberg J. and Dunn J J (1988) OmpT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification J Bacteriol 170, 1245–1253

    PubMed  CAS  Google Scholar 

  8. White C B, Chen Q., Kenyon G. L, and Babbitt P C (1995) A novel activity of ompT. J Biol Chem 270, 12,990–12,994

    Article  PubMed  CAS  Google Scholar 

  9. Leahy D J, Hendrickson W A, Aukhll I, and Erickson H P (1992) Structure of a fibronectin type III domain from tenascm phased by MAD analysts of the selenomethtonyl protein Science 258, 987–991

    Article  PubMed  CAS  Google Scholar 

  10. Wood W (1966) Host spectficity of DNA produced by Escherichia coli. bacterial mutations affecting the restriction and modification of DNA. J Mol Biol 16, 118–133

    Article  PubMed  CAS  Google Scholar 

  11. Doherty A.J., Ashford S.R., Brannigan J A. and Wigley D.B. (1995) A superior host stram for the over-expression of cloned genes using the T7 promoter based vectors Nucleic Acids Res 23, 2074–2075

    Article  PubMed  CAS  Google Scholar 

  12. Derman A. I., Prinz W. A, Belin D, and Beckwith J (1993) Mutations that allow disulfide bond formation in the cytoplasm of Escherichia coli Science 262, 1744–1747

    Article  PubMed  CAS  Google Scholar 

  13. Studier F. W. (1991) Use of bacteriophage T7 lysozyme to improve an inductble T7 expression system. J. Mol Biol 219, 37–44.

    Article  PubMed  CAS  Google Scholar 

  14. Moffatt B. A. and Studier F. W. (1987) T7 lysozyme inhibits transcription by T7 RNA polymerase Cell 49, 221–227

    Article  PubMed  CAS  Google Scholar 

  15. Inouye M., Arnheim N., and Sternglanz R. (1973) Bacteriophage T7 lysozyme is an N-acetylmuramyl-t,-alanme amidase. J Biol Chem. 248, 7247–7252

    PubMed  CAS  Google Scholar 

  16. Chang A C Y and Cohen S N (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmld. J. Bacterial 134, 1141–1156

    CAS  Google Scholar 

  17. Dunn J. J. and Studier F. W (1983) Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. J. Mol Biol 166, 477–535 and erratum (1984) J. Mol. Biol. 175, 111, 112

    Article  PubMed  CAS  Google Scholar 

  18. McAllister W. T., Morris C., Rosenberg A. H, and Studier F. W (1981) Utilization of bacteriophage T7 late promoters in recombinant plasmids during infecnon. J Mol. Biol 153, 527–544.

    Article  PubMed  CAS  Google Scholar 

  19. Sambrook J., Fritsch E. F., and Mamatis T. (1989) Molecular Cloning A Laboratory Manual(2nd ed.), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

    Google Scholar 

  20. Schein C. H. and Noteborn M. H. M. (1989) Production of soluble recombinant proteins in bacteria Bio/Technology 7, 1141–1148

    CAS  Google Scholar 

  21. Ausubel F M., Brent R., Kingston R. E, Moore D. D, Setdman J. G., Smith J. A., and Struhl K. (1989) Expression and purification of maltose binding protein fusions, in Current Protocols in Molecular Biology (Riggs P, ed ), Wiley, New York, pp 16 6 1–16 6 14

    Google Scholar 

  22. Mierendorf R., Yaeger K, and Novy R (1994) The pET system your choice for expression. Innovations 1, 1–3.

    Google Scholar 

  23. Novy R., Berg J., Yaeger K, and Mierendorf R. (1995) pET TRX fusion system for Increased solubility of target proteins expressed in E coli. inNovations 3, 7–9

    Google Scholar 

  24. LaValhe E R, DiBlasio E A, Kovacrc S., Grant K L, Schendel P. F., and McCoy J. M. (1993) A thtoredoxm gene fusion expression system that ctrcumvents inclusion body formation in the E colz cytoplasm. Bio/Technology 11, 187–193

    Article  Google Scholar 

  25. Wickner W., Driessen A. J in, and Hartl F.-U (1991) The enzymology of protein translocation across the Escherwhia coli plasma membrane Ann Rev Biochem 60, 101–124

    Article  PubMed  CAS  Google Scholar 

  26. Hirel P-H, Schmitter J.-M, Dessen P, Fayat G, and Blanquet S (1989) Extent of N-terminal methlonine excision from Escherichia coli proteins is governed by the side-chain length of the penultimate amino acid Proc Natl Acad. Sci. USA 86, 8247–8251.

    Article  PubMed  CAS  Google Scholar 

  27. Lathrop B K., Burack W. R., Biltonen R. L., and Rule G. S. (1992) Expression of a group II phosphohpase A2 from the venom of Agkistrodon piscivorus in Escherichia coli: recovery and renaturation from bacterial inclusion bodies Prot.Exp Purif. 3, 512–517

    Article  CAS  Google Scholar 

  28. Tobtas J W., Shrader T. E, Recap G., and Varchavsky A. (1991) The N-end rule in bacteria. Science 254, 1374–1377.

    Article  Google Scholar 

  29. Pretbtsch G, Ishrhara H., Trtpter D., and Lemeweber M. (1988) Translational controls. Unexpected translation initiation within the coding region of eukaryotic genes expressed in Escherichia coli. Gene 72, 179–186

    Article  Google Scholar 

  30. Halling S. M. and Smith S (1985) Expression in Eschericia coli of multiple products from a chlmeric gene fusion evidence for the presence of procaryotic translational control regions within eucaryotic genes Bio/Technology 3, 715–720.

    Article  CAS  Google Scholar 

  31. Kim J.-S. and Rames R. T (1994) Peptlde tags for a dual affinity fusion system Anal Biochem 219, 165,166

    Article  Google Scholar 

  32. McCormick M and Mierendorf R. (1994) S Tag a multipurpose fusion peptide for recombinant proteins inNovations 1, 4–6

    Google Scholar 

  33. Tessler L-H, Sondermeyer P, Faure T, Dreyer D, Benavente A., Vllleval D, Courtney M, and Lecocq J-P (1984) The influence of mRNA secondary strucure on human IFN-Γ gene expresslon in E coli. Nucleic Acids Res 12,7663–7675

    Article  Google Scholar 

  34. Looman A C, Bodlaender J, De Gruyter M, Vogelaar A, and Van Kmppenberg P H (1986) Secondary structure as a primary determinant of the efficiency of ribosomal binding sites in Escherichia coli. Nucleic Acids Res 14,5481–5496.

    Article  PubMed  CAS  Google Scholar 

  35. Lee N, Zhang S-Q, Cozzitorto J, Yang J-S, and Testa D (1987) Modlfication of mRNA secondary structure and alteration of the expression of human interferon α in Escherichia coli Gene 58, 77–86

    Article  PubMed  CAS  Google Scholar 

  36. Zhang S, Zubay G, and Goldman E (1991) Low-usage codons in Escherichia coli, yeast, fruit flies and primates Gene 105, 61–72

    Article  PubMed  CAS  Google Scholar 

  37. Sorensen M A, Kurland C G, and Pedersen S (1989) Codon usage determines translation rate in Escherichia coli J Mol Biol 207, 365–377

    Article  PubMed  CAS  Google Scholar 

  38. Chen G-F T and 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 

  39. Ikemura T. (1985) Codon usage and tRNA content in unicellular and multlcellu lar organisins. Mol Biol Evol 2, 13–34

    PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Novagen, Madison, WI

    Robert C. Mierendorf, Barbara B. Morris, Beth Hammer & Robert E. Novy

Authors
  1. Robert C. Mierendorf
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  2. Barbara B. Morris
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  3. Beth Hammer
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  4. Robert E. Novy
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Editors and Affiliations

  1. University of Regensburg, Germany

    Udo Reischl

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© 1998 Humana Press Inc.

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Mierendorf, R.C., Morris, B.B., Hammer, B., Novy, R.E. (1998). Expression and Purification of Recombinant Proteins Using the pET System. In: Reischl, U. (eds) Molecular Diagnosis of Infectious Diseases. Methods in Molecular Medicine™, vol 13. Humana Press, Totowa, NJ. https://doi.org/10.1385/0-89603-485-2:257

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  • DOI: https://doi.org/10.1385/0-89603-485-2:257

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-0-89603-485-3

  • Online ISBN: 978-1-59259-597-6

  • eBook Packages: Springer Protocols

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