Molecular events in the processing of recombinant type 1 pre-pro-transforming growth factor beta to the mature polypeptide.
Recently, the simian type 1 transforming growth factor beta (TGF-beta 1) cDNA was expressed at high levels in Chinese hamster ovary (CHO) cells by dihydrofolate reductase-induced gene amplification (L.E. Gentry, N.R. Webb, G.J. Lim, A.M. Brunner, J.E. Ranchalis, D.R. Twardzik, M.N. Lioubin, H. Marquardt, and A.F. Purchio, Mol. Cell. Biol. 7:3418-3427, 1987). We have now purified and characterized the recombinant proteins released by these cells. Analyses of the precursor proteins by amino acid sequencing identified potentially important proteolytic processing sites. Signal peptide cleavage occurs at the Gly-29-Leu-30 peptide bond of pre-pro-TGF-beta 1, yielding pro-TGF-beta 1 (30 to 390). In addition, proteolytic processing of the precursor to yield mature TGF-beta 1 occurs at the dibasic cleavage site immediately preceding Ala-279, indicating that CHO cells possess the appropriate processing enzyme. Greater than 95% of the biological activity detected in the conditioned medium of the CHO transfectant was due to mature, properly processed growth factor. Highly purified recombinant TGF-beta 1 had the same specific biological activity as natural TGF-beta 1. The concentration of TGF-beta 1 required for half-maximal inhibition of Mv1Lu mink lung epithelial cell growth was approximately 1 to 2 pM. Purified precursor inhibited mink lung cell proliferation at 50 to 60 pM concentrations. The purified precursor preparation was shown to consist of pro-TGF-beta 1 (30 to 390), the pro region of the precursor (30 to 278), and mature TGF-beta 1 (279 to 390) interlinked by at least one disulfide bond with the pro portion of the precursor. These recombinant forms of TGF-beta1 should prove useful for further structural and functional studies.
Gentry
L E
LE
Oncogen, Seattle, Washington 98121.
Lioubin
M N
MN
Purchio
A F
AF
Marquardt
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001681:Biological Assay; D002460:Cell Line; D006224:Cricetinae; D004220:Disulfides; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000302:isolation & purification / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D015291:Transforming Growth Factors / Q000302:isolation & purification / Q000378:metabolism*
D004220:Disulfides; D011498:Protein Precursors; D011994:Recombinant Proteins; D015291:Transforming Growth Factors