Effects of unpaired cysteines on yield, solubility and activity of different recombinant antibody constructs expressed in E. coli

New E. coli vectors based on the pOPE/pSTE vector system [Gene 128 (1993) 9 7] were constructed to express a single-chain Fv antibody fragment (scFv), a scFv-streptavidin fusion protein and two disulfide bond-stabilized Fv ant ibody fragments (dsFvs) utilizing different side chain positions for disulf ide stabilization. All of these constructs encoded fusion proteins carrying five C-terminal histidine residues preceded by an unpaired cysteine. The i nfluence of this cysteine, which was originally introduced to allow the che mical modification of the fusion proteins, was assessed by exchanging the t wo amino acids CysIle in front of the carboxy terminal His-tag to SerHis in all constructs. Yield and antigen-binding activity of the antibody constru cts were compared after standard lab-scale periplasmic expression in Escher ichia coli. The removal of the unpaired cysteine resulted in a significant increase in antigen-binding activity of the crude periplasmic extracts. Fur ther, a three-five fold increase or. yield and a significantly improved pur ity were observed after immobilized metal affinity chromatography (IMAC) wi th all four constructs. (C) 2000 Elsevier Science BN. All rights reserved.