Separation, purification, and sequence identification of TGF-beta 1 and TGF-beta 2 from bovine milk.
Cox and Bürk (Eur. J. Biochem., 1991) reported the partial characterization of Milk Growth Factor (MGF) which stimulated the migration of fibroblasts. We have fractionated the partially purified sample by RP-HPLC and obtained the separation of two peaks of activity. The two active components were isolated as pure MGF-a and MGF-b by RP-HPLC and preparative SDS-PAGE. The purified MGF-a, consisting of a single band by gel electrophoresis and a single peak on an HPLC reversed-phase C-4 column, has the same specific activity as TGF-beta 2 in the fibroblast migration assay. MGF-a was digested by endoprotease Asp-N and the cleaved peptides were analyzed by Edman degradation and plasma desorption mass spectrometry (PDMS). The whole sequence of MGF-a determined by automated sequenator and PDMS of S-pyridylethylated protein and selected fragments was found to be identical to that of TGF-beta 2. MGF-b protein mixture separated by SDS-PAGE was electrophoretically transferred onto a Biometra Glassybond membrane, and the blotted MGF-b protein was directly sequenced on an automated sequenator. The identified 29 amino acids sequence of MGF-b was identical to the amino-terminal sequence of TGF-beta 1. Our study demonstrates that MGF is composed of both TGF-beta 1 and TGF-beta 2. TGF-beta 2 (85%) is the predominant form.
Jin
Y
Y
Department of Biotechnology, Ciba-Geigy Ltd., Basel, Switzerland.
Cox
D A
DA
Knecht
R
R
Raschdorf
F
F
Cerletti
N
N
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006133:Growth Substances / Q000737:chemistry / Q000302:isolation & purification*; D008892:Milk / Q000032:analysis*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D012689:Sequence Homology, Nucleic Acid; D016212:Transforming Growth Factor beta / Q000737:chemistry / Q000302:isolation & purification*
D006133:Growth Substances; D016212:Transforming Growth Factor beta