Synthesis and expression of a DNA encoding the Fv domain of an anti-lysozyme monoclonal antibody, HyHEL10, in Streptomyces lividans.
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 (SPssi) in an SPssi::VH-SPssi::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 microgram/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.
Ueda
Y
Y
Department of Industrial Chemistry, Faculty of Engineering, University of Tokyo, Japan.
Tsumoto
K
K
Watanabe
K
K
Kumagai
I
I
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D015870:Gene Expression; D005803:Genes, Immunoglobulin; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D008969:Molecular Sequence Data; D009113:Muramidase / Q000276:immunology; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals; D013302:Streptomyces / Q000235:genetics
D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D009838:Oligodeoxyribonucleotides; D021382:Protein Sorting Signals; D009113:Muramidase