Biosynthetic Antibody Binding Sites: Development of a Single-chain Fv Model Based on Antidinitrophenol IgA Myeloma MOPC 315

Overview

Journal J Protein Chem

Specialties Biochemistry
Chemistry

Date 1991 Dec 1

PMID 1815591

Citations 5

Authors

J E McCartney

L Lederman

E A Drier

G M Wu

R S Batorsky

J S Huston

H Oppermann

Affiliations

Soon will be listed here.

Abstract

The functional antigen binding region of antidinitrophenol mouse IgA myeloma MOPC 315 has been produced as a single-chain Fv (sFv) protein in E. coli. Recombinant 315 proteins included sFv alone, a bifunctional fusion protein with amino-terminal fragment B (FB) of staphylococcal protein A, and a two-chain 315 Fv fragment. Successful refolding of the 315 sFv required formation of disulfide bonds while the polypeptide was in a denatured state, as previously observed for the parent Fv fragment. Affinity-purified recombinant 315 proteins showed full recovery of specific activity, with values for Ka,app of 1.5 to 2.2 x 10(6) M-1, equivalent to the parent 315 Fv fragment. As observed for natural 315 Fv, the sFv region of active FB-sFv315 fusion protein was resistant to pepsin treatment, whereas inactive protein was readily degraded. These experiments will allow the application of protein engineering to the 315 single-chain Fv; such studies can advance structure-function studies of antibody combining sites and lead to an improved understanding of single-chain Fv proteins.

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