title: N-terminal cysteinyl proteins can be prepared using thrombin cleavage.
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 alpha-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.
authors.affiliation: New York Structural Biology Center, 89 Convent Avenue, New York, NY 10027, USA.
authors.forename: Dongsheng
authors.initials: D
authors.lastname: Liu
authors.forename: Rong
authors.initials: R
authors.lastname: Xu
authors.forename: Kaushik
authors.initials: K
authors.lastname: Dutta
authors.forename: David
authors.initials: D
authors.lastname: Cowburn
mesh_terms: D003545:Cysteine / Q000737:chemistry*; D007202:Indicators and Reagents; D015202:Protein Engineering / Q000379:methods*; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism; D013917:Thrombin
chemical_list: D007202:Indicators and Reagents; D011993:Recombinant Fusion Proteins; D011505:Protein-Tyrosine Kinases; D013917:Thrombin; D003545:Cysteine