VARIABLE REGION SEQUENCE MODULATES PERIPLASMIC EXPORT OF A SINGLE-CHAIN FV ANTIBODY FRAGMENT IN ESCHERICHIA-COLI

Using PCR, we have cloned antibody heavy and light chain variable regi on (VH and VL) coding sequences specific far a recombinant hepatitis B virus surface antigen (HBsAg) and assembled these for expression as s ingle-chain Fv (scFv) fragments in Escherichia coli periplasm using th e ompA signal peptide. The vectors also encoded N- or C-terminal Hiss extensions to allow for the purification of the expressed proteins usi ng immobilized metal affinity chromatography (IMAC). We found that the VH-linker-VL configuration of the scFv was not exported to the peripl asm but remained associated with cellular insoluble material, from whi ch it could be extracted, renatured to its active form by gentle dialy sis and purified using IMAC. The molecular size of the scFv suggests t hat the ompA signal peptide was not processed. Based on previous repor ts, we hypothesized that the arginine in framework 1 (FR1) of the VH m ight interfere with translocation To the periplasm by means of the sig nal peptide. Because no arginines are present in FR1 of VL, we reverse d the order of the V-regions in the scFv and observed efficient export of the active scFv to the periplasm. Furthermore, when the arginine i n FR1 of VH was mutated to glycine in the original VH-linker-VL constr uct, active scFv was also exported to the periplasm. Thus, exposed pos itive charges near the signal peptide may account for at least some of the often-encountered difficulties in bacterial scFv expression.