High-throughput production of recombinant antigens for mouse KIAA proteins in Escherichia coli: computational allocation of possible antigenic regions, and construction of expression plasmids of glutathione-S-transferase-fused antigens by an in vitro recombination-assisted method.
Since the end of 2001, we have conducted a project to isolate and determine entire sequences of mouse cDNA clones which encode the polypeptides corresponding to human KIAA proteins. Towards the ultimate goal of this project to clarify the biological functions of KIAA genes, we have set production of antibodies against mouse KIAA gene products based on their sequence information as the next important stage. As the first step, we developed a high-throughput system utilizing shotgun clones generated during entire sequencing of mouse KIAA cDNAs. The system consists of the following three parts: (1) Shotgun clones encoding regions suitable for production of antigens were selected using a newly developed browser system; (2) the protein-coding sequences of the selected shotgun clones were transferred into an expression vector by in vitro recombination-assisted method in a 96-well format, and expressed as glutathione S-transferase fusion proteins in Escherichia coli; and (3) the solubility of the recombinant antigens were preliminarily assessed in a small-scale culture and then large-scale production and purification was performed using glutathione-affinity beads or retrieval from polyacrylamide gels depending on their solubility. Using these systems, we successfully produced and purified 400 antigens for production of mKIAA antibodies to date.
Hara
Yasuhiro
Y
Chiba Industry Advancement Center, 2-6 Nakase, Mihama-ku, Chiba 261-7126, Japan.
Shimada
Kiyo
K
Kohga
Hiroshi
H
Ohara
Osamu
O
Koga
Hisashi
H
D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000378:metabolism; D000941:Antigens / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D010957:Plasmids; D011506:Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D011995:Recombination, Genetic
D000906:Antibodies; D000941:Antigens; D003470:Culture Media; D011506:Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase