A novel recombinant system for functional expression of myonecrotic snake phospholipase A2 in Escherichia coli using a new fusion affinity tag

Minae Seto, Tomohisa Ogawa, Kyousuke Kodama, Koji Muramoto, Yoshitaka Kanayama, Yasuo Sakai, Takahito Chijiwa, Motonori Ohno

Research output: Contribution to journalArticlepeer-review

7 Citations (Scopus)

Abstract

A novel recombinant expression system in Escherichia coli was developed using conger eel galectin, namely, congerin II, as an affinity tag. This system was applied for the functional expression of myotoxic lysine-49-phospholipase A2 ([Lys49]PLA2), termed BPII and obtained from Protobothrops flavoviridis (Pf) venom. Recombinant Pf BPII fused with a congerin tag has been successfully expressed as a soluble fraction and showed better quantitative yield when folded correctly. The solubility of the recombinant congerin II-tagged BPII increased up to >90% in E. coli strain JM109 when coexpressed with the molecular chaperones GroEL, GroES, and trigger factor (Tf). The tag protein was cleaved by digestion with restriction protease, such as α-thrombin or Microbacterium liquefaciens protease (MLP), to obtain completely active recombinant BPII. Thus, the congerin-tagged fusion systems containing the cleavage recognition site for α-thrombin or MLP were demonstrated to be highly efficient and useful for producing proteins of desired solubility and activity.

Original languageEnglish
Pages (from-to)194-202
Number of pages9
JournalProtein Expression and Purification
Volume58
Issue number2
DOIs
Publication statusPublished - 2008 Apr 1

Keywords

  • Affinity tag
  • Chaperones
  • Congerin
  • Escherichia coli
  • Galectin
  • Microbacterium liquefaciens protease
  • Myotoxin
  • Phospholipase A2
  • Protobothrops flavoviridis
  • Recombinant expression
  • Snake venome
  • Thrombin

ASJC Scopus subject areas

  • Biotechnology

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