A Photobacterium sp. α2-6-sialyltransferase (Psp2,6ST) mutant with an increased expression level and improved activities in sialylating Tn antigens

Li Ding, Chao Zhao, Jingyao Qu, Yanhong Li, Go Sugiarto, Hai Yu, Junru Wang, Xi Chen

Research output: Contribution to journalArticle

14 Scopus citations

Abstract

Abstract In order to improve the catalytic efficiency of recombinant Photobacterium sp. JT-ISH-224 α2-6-sialyltransferase Psp2,6ST(15-501)-His<inf>6</inf> in sialylating α-GalNAc-containing acceptors for the synthesis of tumor-associated carbohydrate antigens sialyl Tn (STn), protein crystal structure-based mutagenesis studies were carried out. Among several mutants obtained by altering the residues close to the acceptor substrate binding pocket, mutant A366G was shown to improve the sialyltransferase activity of Psp2,6ST(15-501)-His<inf>6</inf> toward α-GalNAc-containing acceptors by 21-115% without significantly affecting its sialylation activity to β-galactosides. Furthermore, the expression level was improved from 18-40 mg L<sup>-1</sup> for the wild-type enzyme to 72-110 mg L<sup>-1</sup> for the A366G mutant. In situ generation of CMP-sialic acid in a one-pot two-enzyme system was shown effective in overcoming the high donor hydrolysis of the enzyme. Mutant A366G performed better than the wild-type Psp2,6ST(15-501)-His<inf>6</inf> for synthesizing Neu5Acα2-6GalNAcαOSer/Thr STn antigens.

Original languageEnglish (US)
Article number6909
Pages (from-to)127-133
Number of pages7
JournalCarbohydrate Research
Volume408
DOIs
StatePublished - May 18 2015

Keywords

  • Improved protein expression
  • Mutagenesis
  • Sialyltransferase
  • Sialyltransferase mutant
  • STn antigen

ASJC Scopus subject areas

  • Biochemistry
  • Analytical Chemistry
  • Organic Chemistry

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