Production of recombinant notechis 11'2L, an enzymatically active mutant of a phospholipase A2 from Notechis scutatus scutatus venom, as directly generated by cleavage of a fusion protein produced in Escherichia coli.
We have constructed an expression vector to produce, in Escherichia coli, a fusion protein containing successively two IgG binding domains from staphyloccocal protein A, a nine-amino-acid linker peptide terminating in a methionine residue and the phospholipase A2 notechis 11'2L, an isoform of notexin of Notechis scutatus scutatus venom. Notechis 11'2L is a mutant of the naturally occurring notechis 11'2 [Bouchier, C., Boyot, P., Tesson, F., Trémeau, O., Bouet, F., Hodgson, D., Boulain, J. C. & Ménez, A. (1991) Eur. J. Biochem. 202, 493-500] in which Met8 has been replaced by Leu. The fusion protein was recovered in the periplasmic extract with a yield of 0.25 mg/l culture. It was hydrolyzed with cyanogen bromide, yielding a protein having the molecular mass, amino acid composition and N-terminal sequence of notechis 11'2L. Notechis 11'2L and the wild notechis 11'2 displayed identical circular dichroic spectra and shared similar enzymatic, myotoxic and antigenic properties, suggesting that the recombinant notechis 11'2L was directly generated in a correctly folded form.
Hodgson
D
D
Département d'Ingéniérie et d'Etudes des Protéines, CE Saclay, Gif-sur-Yvette, France.
Gasparini
S
S
Drevet
P
P
Ducancel
F
F
Bouet
F
F
Boulain
J C
JC
Harris
J B
JB
Menez
A
A
D001483:Base Sequence; D004546:Elapid Venoms / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010741:Phospholipases A / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D054467:Phospholipases A2; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*
D004546:Elapid Venoms; D011993:Recombinant Fusion Proteins; C072139:Notechis 11'2; D010741:Phospholipases A; D054467:Phospholipases A2