CLONING AND EXPRESSION OF THE VARIABLE REGIONS OF MOUSE MYELOMA PROTEIN MOPC315 IN ESCHERICHIA-COLI - RECOVERY OF ACTIVE FV FRAGMENTS

Recombinant DNA techniques were used to clone and express the F(v) portion of MOPC315, a mouse myeloma protein with a high affinity for 2,4-dinitrophenyl (DNP). The F(v) fragment consists of a heterodimer of heavy and light chain variable domains (V(H) and V(L)). Two separate bacterial plasmid constructs, containing either a variable region cDNA for the light chain or a variable region synthetic gene for the heavy chain demonstrated high levels of expression (150-200 mg/L) under control of the bacteriophage T7 promoter. Recombinant chains were initially recovered as inclusion bodies and then dissolved separately in 8 M urea, combined together, and refolded by subsequent chaotrope removal. Biologically active F(V) was affinity purified from the chain mixture by specific binding to DNP-lysine-Sepharose. Yields of active material as high as 20% were obtained with activity confirmed by fluorescence quench analysis. The purified F(V)s composed of recombinant and native chain mixtures yielded similar results. Recombinant MOPC315 F(V) activity was also obtained using a single chain construct (sF(V)), in which recombinant V(H) and V(L) were linked via a (Gly4Ser)3 spacer region. Binding affinity of the sF(V) was shown to be the same as the recombinant and native F(V)s. The ease of purification and characterization of active MOPC315 F(V) makes this system useful in the study of the optimization of antibody production in bacteria.