PMID:18207418
| Citation |
Seto, M, Ogawa, T, Kodama, K, Muramoto, K, Kanayama, Y, Sakai, Y, Chijiwa, T and Ohno, M (2008) A novel recombinant system for functional expression of myonecrotic snake phospholipase A(2) in Escherichia coli using a new fusion affinity tag. Protein Expr. Purif. 58:194-202 |
|---|---|
| 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 A(2) ([Lys(49)]PLA(2)), 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 alpha-thrombin or Microbacterium liquefaciens protease (MLP), to obtain completely active recombinant BPII. Thus, the congerin-tagged fusion systems containing the cleavage recognition site for alpha-thrombin or MLP were demonstrated to be highly efficient and useful for producing proteins of desired solubility and activity. |
| Links |
PubMed Online version:10.1016/j.pep.2007.11.013 |
| Keywords |
Actinomycetales/enzymology; Crotalid Venoms/enzymology; Escherichia coli/metabolism; Galectins/chemistry; Peptide Hydrolases/metabolism; Phospholipases A2/biosynthesis; Phospholipases A2/isolation & purification; Phospholipases A2/metabolism; Recombinant Fusion Proteins/biosynthesis; Recombinant Fusion Proteins/isolation & purification; Thrombin/metabolism |
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