Bacterial expression and purification of the Fab fragment of a monoclonal antibody specific for the low-density lipoprotein receptor-binding site of human apolipoprotein E

We report the bacterial expression and the purification of a monoclonal ant ibody (mAb) specific for an epitope that coincides with the LDL receptor (L DLr)binding domain of human apolipoprotein E (apoE), This antibody resemble s the LDLr in its primary structure and its specificity for apoE variants. The recombinant Fab (rFab) fragment of mAb 2E8, consisting of the entire li ght chain and the Fd portion of the heavy chain, was expressed in Escherich ia coli and purified to homogeneity. Purification was facilitated by includ ing a five-histidine carboxyl-terminal extension on the Fd chain. A 5- to 1 0-fold difference in yield of the antibody was observed when the plasmid wa s expressed in two different strains of E. coli, Typically 2-6 mg of rFab p er liter of culture medium was recovered in the periplasm of the TG1 strain and less than 1 mg was recovered in the periplasm of the XL1-Blue strain. Culture temperatures above 35 degrees C or inclusion of sucrose in the medi um reduced rFab yields. The 2E8 rFab was indistinguishable from Fab prepare d from 2E8 hybridoma-generated IgG with respect to its affinity and fine sp ecificity. We are using this system to express a panel of 2E8 variant Fabs that will be used as probes to establish the structural features responsibl e for the binding of apoE to the LDLr. (C) 1999 Academic Press.