Production of a functional anti-scorpion hemocyanin scFv in Escherichia coli.

Abstract:

:A58 is a murine monoclonal antibody raised against the internal dimeric subunit A alpha 1 of the hemocyanin of the scorpion Androctonus australis. When A58 is tested against a range of related hemocyanins, it exhibits cross-reactivity with a limited number of scorpion species. All of them belong to a single Scorpion Family: the Buthidae. We have utilized a bacterial expression-export system in which the variable regions of A58 are expressed as a single-chain variable fragment (scFv). Polymerase chain reaction (PCR) was used for the cloning and modification of the heavy and light variable regions which were assembled into a scFv. After insertion into a vector which contains the pectate lyase signal sequence from Erwinia caratovora for periplasmic expression, a scFv protein was produced in high yield as a soluble and functional protein. The bacterial produced A58 scFv has binding properties similar to those of the original murine antibody. It binds specifically the heterodimeric subunit of Androctonus australis hemocyanin, cross-reacts with only one subunit of each Buthidae investigated, and blocks the recognition of the hemocyanin by antibody A58 in competitive enzyme-linked immunosorbant assay. Previous reports have demonstrated the value of monoclonal antibodies in taxonomical and molecular evolution studies. A58scFv is the first of a new generation of antigen-binding proteins which should prove useful both in taxonomical studies and in the analysis of VH--VL structure-function relationships of antibodies.

journal_name

Arch Biochem Biophys

authors

Billiald P,Motta G,Vaux DJ

doi

10.1006/abbi.1995.1185

subject

Has Abstract

pub_date

1995-03-10 00:00:00

pages

429-38

issue

2

eissn

0003-9861

issn

1096-0384

pii

S0003-9861(85)71185-X

journal_volume

317

pub_type

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