Generation of recombinant human large latent transforming growth factor-beta 1 and monoclonal antibodies to it.
Transforming growth factor beta 1 (TGF-beta 1) is a regulator of cell growth and differentiation. It is produced in various of cells and tissues as a biologically latent complex, whose significance is still unknown. We established a Chinese hamster ovary cells that produced recombinant human large latent TGF-beta 1. The growth factor was purified from serum-free conditioned medium of the cell line was purified to apparent homogeneity by four steps of column chromatography. The purified protein gave a single band with the apparent molecular weight of 210,000 on SDS-PAGE, and had four subunits, of 12.5, 40, 53, and 150-190 kDa. These components were identical to TGF-beta 1, the N-terminal remnant of pro-TGF-beta 1, pro-TGF-beta 1, and latent TGF-beta 1 binding protein, respectively. The purified growth factor had biological activity similar to that of the growth factor purified from human platelets. We prepared four monoclonal antibodies by immunization of mice with the recombinant protein. In western blotting, two of the antibodies bound to latent TGF-beta 1 binding protein. The two other antibodies reacted with the N-terminal remnant of pro-TGF-beta 1. Recombinant large latent TGF-beta 1 and its monoclonal antibodies could be used for detailed structural and functional studies of the large latent TGF-beta 1 complex.
Tsumura
H
H
Department of Biology, Faculty of Science, Niigata University, Japan. htsumura@kirin.co.jp
Ishii
Y
Y
Shimizu
E
E
Ohashi
H
H
Mori
K J
KJ
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D016466:CHO Cells; D002850:Chromatography, Gel; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000276:immunology; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000276:immunology*
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta