Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase - PubMed

Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase

K L Guan et al. Anal Biochem. 1991.

Abstract

Several systems have been developed to allow for rapid and efficient purification of recombinant proteins expressed in bacteria. The expression of polypeptides in frame with glutathione S-transferase (GST) allows for purification of the fusion proteins from crude bacterial extracts under nondenaturing conditions by affinity chromatography on glutathione agarose (D. B. Smith and K. S. Johnson, 1988, Gene 67, 31-40). This vector expression system has also incorporated specific protease cleavage sites to facilitate proteolysis of the bacterial fusion proteins. In our hands, the cleavage of these fusion proteins at a thrombin cleavage site proceeded slowly. To facilitate the cleavage of fusion proteins, we have introduced a glycine-rich linker (glycine kinker) containing the sequence P.G.I.S.G.G.G.G.G located immediately following the thrombin cleavage site. This glycine kinker greatly increases the thrombin cleavage efficiency of several fusion proteins. The introduction of the glycine kinker into fusion proteins allows for the cleavage of the fusion proteins while they are attached to the affinity resin resulting in a single step purification of the recombinant protein. More than 2 mg of the highly purified protein was obtained from the equivalent of 100 ml of bacterial culture within a few hours when a protein tyrosine phosphatase was employed as a test protein. The vector, pGEX-KG, has also been modified to facilitate cloning of a variety of cDNAs in all reading frames and has been successfully used to express several eukaryotic proteins.

Similar articles

• New vectors for high level expression of recombinant proteins in bacteria.

  Hakes DJ, Dixon JE. Hakes DJ, et al. Anal Biochem. 1992 May 1;202(2):293-8. doi: 10.1016/0003-2697(92)90108-j. Anal Biochem. 1992. PMID: 1519755

• Ligation-independent cloning of glutathione S-transferase fusion genes for expression in Escherichia coli.

  Haun RS, Moss J. Haun RS, et al. Gene. 1992 Mar 1;112(1):37-43. doi: 10.1016/0378-1119(92)90300-e. Gene. 1992. PMID: 1339364

• Expression and thrombin cleavage of Poa p IX recombinant allergens fused to glutathione S-transferase.

  Olsen E, Mohapatra SS. Olsen E, et al. Int Arch Allergy Immunol. 1992;98(4):343-8. doi: 10.1159/000236209. Int Arch Allergy Immunol. 1992. PMID: 1422262

• A critical review of the methods for cleavage of fusion proteins with thrombin and factor Xa.

  Jenny RJ, Mann KG, Lundblad RL. Jenny RJ, et al. Protein Expr Purif. 2003 Sep;31(1):1-11. doi: 10.1016/s1046-5928(03)00168-2. Protein Expr Purif. 2003. PMID: 12963335 Review.

• Targeted expression, purification, and cleavage of fusion proteins from inclusion bodies in Escherichia coli.

  Hwang PM, Pan JS, Sykes BD. Hwang PM, et al. FEBS Lett. 2014 Jan 21;588(2):247-52. doi: 10.1016/j.febslet.2013.09.028. Epub 2013 Sep 27. FEBS Lett. 2014. PMID: 24076468 Review.

Cited by

• X-linked intellectual disability gene CASK regulates postnatal brain growth in a non-cell autonomous manner.

  Srivastava S, McMillan R, Willis J, Clark H, Chavan V, Liang C, Zhang H, Hulver M, Mukherjee K. Srivastava S, et al. Acta Neuropathol Commun. 2016 Mar 31;4:30. doi: 10.1186/s40478-016-0295-6. Acta Neuropathol Commun. 2016. PMID: 27036546 Free PMC article.

• The Arabidopsis homolog of the mammalian OS-9 protein plays a key role in the endoplasmic reticulum-associated degradation of misfolded receptor-like kinases.

  Su W, Liu Y, Xia Y, Hong Z, Li J. Su W, et al. Mol Plant. 2012 Jul;5(4):929-40. doi: 10.1093/mp/sss042. Epub 2012 Apr 19. Mol Plant. 2012. PMID: 22516478 Free PMC article.

• Munc18a does not alter fusion rates mediated by neuronal SNAREs, synaptotagmin, and complexin.

  Zhang Y, Diao J, Colbert KN, Lai Y, Pfuetzner RA, Padolina MS, Vivona S, Ressl S, Cipriano DJ, Choi UB, Shah N, Weis WI, Brunger AT. Zhang Y, et al. J Biol Chem. 2015 Apr 17;290(16):10518-34. doi: 10.1074/jbc.M114.630772. Epub 2015 Feb 25. J Biol Chem. 2015. PMID: 25716318 Free PMC article.

• The mechanism of complex formation between Fli-1 and SRF transcription factors.

  Dalgleish P, Sharrocks AD. Dalgleish P, et al. Nucleic Acids Res. 2000 Jan 15;28(2):560-9. doi: 10.1093/nar/28.2.560. Nucleic Acids Res. 2000. PMID: 10606656 Free PMC article.

• Kaposi's sarcoma-associated herpesvirus LANA2 is a B-cell-specific latent viral protein that inhibits p53.

  Rivas C, Thlick AE, Parravicini C, Moore PS, Chang Y. Rivas C, et al. J Virol. 2001 Jan;75(1):429-38. doi: 10.1128/JVI.75.1.429-438.2001. J Virol. 2001. PMID: 11119611 Free PMC article.

Publication types

MeSH terms

Substances

LinkOut - more resources

• Full Text Sources

  • Elsevier Science

• Other Literature Sources

  • The Lens - Patent Citations Database

• Research Materials

  • NCI CPTC Antibody Characterization Program