Expression and purification of the full-length N-acetylgalactosaminyltransferase and galactosyltransferase from Campylobacter jejuni in Escherichia coli

Jong Min Yang, Gi Eob Kim, Kyeong Rok Kim, Chang Sup Kim

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

The successful enzymatic synthesis of various ganglioside-related oligosaccharides requires many available glycan-processing enzymes. However, the number of available glycan-processing enzymes remains limited. In this study, the full-length CgtA43456 (β-(1→4)-N-acetylgalactosaminyltransferase) and CgtB11168 (β-(1→3)-galactosyltransferase) were successfully produced from Escherichia coli through the optimization of E. coli–preferable codon usage, selection of E. coli strain, and use of the molecular chaperone GroEL-GroES (GroEL/ES). The CgtA43456 enzyme was produced as a soluble form in E. coli C41(DE3) co-expressed with codon-optimized CgtA43456 and GroEL/ES. However, soluble CgtB11168 was well expressed in E. coli C41(DE3) with only the codon-optimized CgtB11168. Rather, when co-expressed with GroEL/ES, total production of CgtB11168 was reduced. Using immobilized-metal affinity chromatography, the CgtA43456 and CgtB11168 proteins were obtained with approximately 75–78 % purity. The purified CgtA43456 showed a specific activity of 21 mU/mg using UDP-N-acetylgalactosamine and GM3 trisaccharide as donor and acceptor, respectively. The purified CgtB11168 catalyzed the transfer of galactose from UDP-Gal to GM2 tetrasaccharide with a specific activity of 16 mU/mg. We propose that they could be used as catalysts for enzymatic synthesis of GM1 ganglioside–related oligosaccharides.

Original languageEnglish
Article number109489
JournalEnzyme and Microbial Technology
Volume135
DOIs
StatePublished - Apr 2020

Keywords

  • Campylobacter jejuni
  • Escherichia coli
  • Expression
  • Purification
  • β-(1→3)-Galactosyltransferase
  • β-(1→4)-N-acetylgalactosaminyltransferase

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