Periplasmic Expression and Purification of Recombinant Fabs

Abstract

The ability to refine the affinity, specificity, and immunogenicity of recombinant antibodies (Abs) offers distinct advantages for the preparation of in vivo diagnostic and therapeutic immunoreagents. The bacterial expression vector pComb3 allows production of recombinant Ab Fabs (rFab) (1). Light-chain (LC) and heavy-chain (HC) Ab cDNA fragments are inserted separately into this vector, which produces heterodimeric rFab Abs that are isolated in a native form. Fabs generated with the pComb3 expression vector are documented to bind Ag with affinities similar to that of the parental hybridoma-generated Fab (2). In the plasmid, both chains are independently controlled by isopropyl-1-β-D-thioglactopyranoside (IPTG)-inducible lac expression. The original pComb3 vector allows for the display of rFabs on the surface of M13 filamentous phage and can be converted to produce rFabs in a soluble form. We have modified this vector to allow the expression and purification of soluble Fab Ab fragments using immobilized metal-affinity chromatography via a pentahistidine (His) tag fused to the HC constant region 1 Fig. 1; 2). A similar modification to pComb3 (pComb3x) designed exclusively for the expression of soluble rFabs has been performed by Barbas at the Scripps Research Institute (see Note 1).