title: A novel recombinant system for functional expression of myonecrotic snake phospholipase A(2) in Escherichia coli using a new fusion affinity tag.
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.
authors.affiliation: Department of Biomolecular Science, Graduate School of Life Sciences, Tohoku University, 1-1 Tsutsumidori amamiyamachi, Aoba-ku, Sendai 981-8555, Japan.
authors.forename: Minae
authors.initials: M
authors.lastname: Seto
authors.forename: Tomohisa
authors.initials: T
authors.lastname: Ogawa
authors.forename: Kyousuke
authors.initials: K
authors.lastname: Kodama
authors.forename: Koji
authors.initials: K
authors.lastname: Muramoto
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Kanayama
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Sakai
authors.forename: Takahito
authors.initials: T
authors.lastname: Chijiwa
authors.forename: Motonori
authors.initials: M
authors.lastname: Ohno
mesh_terms: D000192:Actinomycetales / Q000201:enzymology; D003435:Crotalid Venoms / Q000201:enzymology*; D004926:Escherichia coli / Q000378:metabolism*; D037161:Galectins / Q000737:chemistry; D010447:Peptide Hydrolases / Q000378:metabolism; D054467:Phospholipases A2 / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013917:Thrombin / Q000378:metabolism
chemical_list: D003435:Crotalid Venoms; D037161:Galectins; D011993:Recombinant Fusion Proteins; C122080:congerin II protein, Conger eel; D054467:Phospholipases A2; D010447:Peptide Hydrolases; D013917:Thrombin