N-terminal cysteinyl proteins can be prepared using thrombin cleavage

Dongsheng Liu, Rong Xu, Kaushik Dutta, David Cowburn

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

Expressed protein ligation - which allows native proteins to be selectively linked together by a normal peptide bond in an aqueous environment - has emerged as a powerful technique. The technique requires the formation of a C-terminal α-thioester and an N-terminal Cys. An N-terminal Cys can be formed by enzymatic cleavage, commonly using the Factor Xa and TEV proteases. We show that thrombin can be used for the formation of N-terminal Cys, providing another choice of reagents for expressed protein ligation. Proteins with N-terminal Cys can be obtained by the convenient modification of vectors with the putative thrombin cleavage site LVPRG to LVPRC. Two example protein domains (Csk and Abl tyrosine kinase domain) with N-terminal Cys are demonstrated using this method. The use of thrombin protease to generate N-terminal Cys overcomes some of the limitations of existing methods, making it generally useful for expressed protein ligation and other biotechnological applications.

Original languageEnglish (US)
Pages (from-to)1163-1167
Number of pages5
JournalFEBS Letters
Volume582
Issue number7
DOIs
StatePublished - Apr 2 2008

Fingerprint

Thrombin
Ligation
Proteins
Factor Xa
Protein-Tyrosine Kinases
Peptide Hydrolases
Peptides

Keywords

  • Cysteine
  • Expressed protein ligation
  • Segmental isotopic labeling
  • Thrombin
  • Tyrosine kinase

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

N-terminal cysteinyl proteins can be prepared using thrombin cleavage. / Liu, Dongsheng; Xu, Rong; Dutta, Kaushik; Cowburn, David.

In: FEBS Letters, Vol. 582, No. 7, 02.04.2008, p. 1163-1167.

Research output: Contribution to journalArticle

Liu, Dongsheng ; Xu, Rong ; Dutta, Kaushik ; Cowburn, David. / N-terminal cysteinyl proteins can be prepared using thrombin cleavage. In: FEBS Letters. 2008 ; Vol. 582, No. 7. pp. 1163-1167.
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