A novel protease obtained from FBS-containing culture supernatant, that processes single chain form hepatocyte growth factor to two chain form in serum-free culture.
The recombinant human hepatocyte growth factor (r-hHGF) produced by Chinese hamster ovary cells transfected with hHGF cDNA (CHO BD-24 cells) was the two chain form in fetal bovine serum (FBS) containing culture. However, in serum-free culture the non-processed r-hHGF, single chain form, was detected with two chain form r-hHGF. We purified the protease that proteolytically processed single chain r-hHGF to two chain form r-hHGF. A protease was purified to give a single peak from the culture supernatant by use of several column chromatographies. When this protease was added to serum-free culture of CHO BD-24 cells, the proteolytic processing of single chain r-hHGF to two chain form r-hHGF was completely achieved. This protease was found to be composed of two peptide chains with molecular mass of 38 kDa under non-reducing condition by SDS-PAGE. The results of N-terminal amino acid sequence analysis and inhibitor selectivity suggested that this protease was a novel serine protease originating from fetal bovine serum.
Biosciences Laboratory, Mitsubishi Kasei Corp., Yokohama, Japan.
T
T
Shimomura
M
M
Ochiai
J
J
Kondo
Y
Y
Morimoto
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D017077:Culture Media, Conditioned / Q000378:metabolism*; D016895:Culture Media, Serum-Free; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D017228:Hepatocyte Growth Factor / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism
D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D017228:Hepatocyte Growth Factor; D010450:Endopeptidases