Skip to main content
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1998 Aug;7(8):1839–1842. doi: 10.1002/pro.5560070821

Overexpression of recombinant proteins with a C-terminal thiocarboxylate: implications for protein semisynthesis and thiamin biosynthesis.

C Kinsland 1, S V Taylor 1, N L Kelleher 1, F W McLafferty 1, T P Begley 1
PMCID: PMC2144084  PMID: 10082383

Abstract

A facile and rapid method for the production of protein C-terminal thiocarboxylates on DNA-encoded polypeptides is described. This method, which relies on the mechanism of the cleavage reaction of intein-containing fusion proteins, can produce multi-milligram quantities of protein C-terminal thiocarboxylate quickly and inexpensively. The utility of this method for protein semisynthesis and implications for studies on the biosynthesis of thiamin are discussed.

Full Text

The Full Text of this article is available as a PDF (1.1 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Begley T. P. The biosynthesis and degradation of thiamin (vitamin B1). Nat Prod Rep. 1996 Jun;13(3):177–185. doi: 10.1039/np9961300177. [DOI] [PubMed] [Google Scholar]
  2. Chong S., Mersha F. B., Comb D. G., Scott M. E., Landry D., Vence L. M., Perler F. B., Benner J., Kucera R. B., Hirvonen C. A. Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element. Gene. 1997 Jun 19;192(2):271–281. doi: 10.1016/s0378-1119(97)00105-4. [DOI] [PubMed] [Google Scholar]
  3. Muir T. W., Dawson P. E., Kent S. B. Protein synthesis by chemical ligation of unprotected peptides in aqueous solution. Methods Enzymol. 1997;289:266–298. doi: 10.1016/s0076-6879(97)89052-0. [DOI] [PubMed] [Google Scholar]
  4. Perler F. B., Xu M. Q., Paulus H. Protein splicing and autoproteolysis mechanisms. Curr Opin Chem Biol. 1997 Oct;1(3):292–299. doi: 10.1016/s1367-5931(97)80065-8. [DOI] [PubMed] [Google Scholar]
  5. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  6. Shao Y., Paulus H. Protein splicing: estimation of the rate of O-N and S-N acyl rearrangements, the last step of the splicing process. J Pept Res. 1997 Sep;50(3):193–198. doi: 10.1111/j.1399-3011.1997.tb01185.x. [DOI] [PubMed] [Google Scholar]
  7. Shao Y., Xu M. Q., Paulus H. Protein splicing: characterization of the aminosuccinimide residue at the carboxyl terminus of the excised intervening sequence. Biochemistry. 1995 Aug 29;34(34):10844–10850. doi: 10.1021/bi00034a017. [DOI] [PubMed] [Google Scholar]
  8. Tam J. P., Lu Y. A., Liu C. F., Shao J. Peptide synthesis using unprotected peptides through orthogonal coupling methods. Proc Natl Acad Sci U S A. 1995 Dec 19;92(26):12485–12489. doi: 10.1073/pnas.92.26.12485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Taylor S. V., Kelleher N. L., Kinsland C., Chiu H. J., Costello C. A., Backstrom A. D., McLafferty F. W., Begley T. P. Thiamin biosynthesis in Escherichia coli. Identification of ThiS thiocarboxylate as the immediate sulfur donor in the thiazole formation. J Biol Chem. 1998 Jun 26;273(26):16555–16560. doi: 10.1074/jbc.273.26.16555. [DOI] [PubMed] [Google Scholar]
  10. Vander Horn P. B., Backstrom A. D., Stewart V., Begley T. P. Structural genes for thiamine biosynthetic enzymes (thiCEFGH) in Escherichia coli K-12. J Bacteriol. 1993 Feb;175(4):982–992. doi: 10.1128/jb.175.4.982-992.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Wilm M., Mann M. Analytical properties of the nanoelectrospray ion source. Anal Chem. 1996 Jan 1;68(1):1–8. doi: 10.1021/ac9509519. [DOI] [PubMed] [Google Scholar]
  12. Xu M. Q., Comb D. G., Paulus H., Noren C. J., Shao Y., Perler F. B. Protein splicing: an analysis of the branched intermediate and its resolution by succinimide formation. EMBO J. 1994 Dec 1;13(23):5517–5522. doi: 10.1002/j.1460-2075.1994.tb06888.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES