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Production of recombinant notechis 11′2L, an enzymatically active mutant of a phospholipase A 2 from Notechis scutatus scutatus venom, as directly generated by cleavage of a fusion protein produced in Escherichia coli

1993; Wiley; Volume: 212; Issue: 2 Linguagem: Inglês

10.1111/j.1432-1033.1993.tb17680.x

1432-1033

Darren Hodgson, Sylvaine Gasparini, Pascal Drevet, Frédéric Ducancel, Françoise Bouet, Jean‐Claude Boulain, John B. Harris, Andre Ménèz,

Nicotinic Acetylcholine Receptors Study

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 A 2 notechis 11′2L, an isoform of notexin of Notechis scutatus scutatus , venom. Notechis 11′2L is a mutant of the naturally occuring 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 J. Biochem. 202 , 493–500] in which Met8 has been replaced by Leu.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/I 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.