Kumagai Izumi

Synthesis and expression of a DNA encoding the Fv domain of an anti-lysozyme monoclonal antibody, HyHEL10, in Streptomyces lividans

Summary A secretory production system for the Fv domain of a monoclonal antibody (mAb) against hen egg-white lysozyme (HEL) was established in Streptomyces lividans using a chemically synthesized gene. The synthetic DNAs encoding the Fv fragments (VH and VL) of the anti-HEL mAb, HyHEL10, were fused to DNA encoding the signal peptide of Streptomyces subtilisin inhibitor (SP ssi ) in an SP ssi :: VH-SP ssi ::VL dicistronic arrangement. The genes were expressed under the control of the ssi promoter using S. lividans as host. Each Fv fragment was accurately processed and secreted into the growth medium. No inclusion bodies were produced. The Fv fragments were isolated from culture supernatant by a two-step purification (affinity chromatography and gel filtration) with a high yield (approx. 1 μg/ml). Purified Fv fragments bound to HEL specifically, and completely inhibited the catalytic activity of HEL at a molar ratio of 1.25 for Fv vs. HEL.

Secretory production of chicken ovomucoid domain 3 by Escherichia coli and alteration of inhibitory specificity toward proteases by substitution of the P1 site residue

Abstract Ovomucoids are commonly present in bird egg white and exhibit inhibitory activity toward various serine proteases. To investigate the structure-function relationship of ovomucoid domain 3, we established a secretory expression system for the chicken ovomucoid domain 3 (OMCHI3)-encoding gene in Escherichia coli by ligating it downstream from the tac promoter and signal peptide of E. coli alkaline phosphatase. E. coli JM105 was transformed with the resulting plasmid and induced with l mM isopropyl-β- d -thiogalactopyranoside (IPTG). The mature OMCHI3 was detected in the culture supernatant, and was purified to homogeneity by three-step chromatography. Amino-acid sequence analysis showed that processing by the signal peptidase was carried out exactly at the expected site. Measurements of circular dichroism spectra and inhibitory activity indicated that OMCHI3 was produced in the properly folded form. Furthermore, site-specific replacement of the Ala residue at the P1 site with Met or Lys resulted in acquisition of inhibitory activity toward chymotrypsin or trypsin, respectively, indicating that the P1 site is the predominant determinant for inhibitory specificity.