Apr 6, 1976

Preparation of Fv fragment from the mouse myeloma XRPC-25 immunoglobulin possessing anti-dinitrophenyl activity

Biochemistry
J Sharon, D Givol

Abstract

The myeloma IgA protein produced by plasmacytoma XRPC-25, was isolated by affinity chromatography on dinitrophenyllysine-Sepharose. The affinity constant of the intact protein or its Fab' toward 2,4-dinitrophenyl-L-lysine (Dnp) was found to be 2.6 X 10(5) M-1. In order to prepare an Fv fragment (Hochman, J., Inbar, D., and Givol, D. (1973), Biochemistry 12, 1130) from this protein, the heavy and light chains were separated and the light chain was digested with trypsin at pH 8.2 to yield half a light chain. This digest was reassociated with the heavy chain and the recombinant was digested with papain at pH 5.7. Fractionation of this digest on a Sephadex G-75 column and Dnp-lysine-Sepharose resulted in the isolation of an Fv fragment which possesses one binding site for Dnplysine (Ka = 2.0 X 10(5) M-1). The active Fv fragment has a molecular weight of 23,400 and is composed of two peptide chains, each having a molecular weight of approximately 12,000. The N-terminal residues of these chains are aspartic and glutamic acids, which are also N-terminal in the heavy and light chains, indicating that the Fv is composed of VL and VH.

  • References11
  • Citations31

References

Mentioned in this Paper

Macromolecular Compounds
Dinitrophenols
Immunoglobulin Activity
Binding Sites, Antibody
Immunoglobulin Fv Fragments
SLC3A2 gene
Digests
Plasma Protein Binding Capacity
Lysine
Immunoglobulins

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