Purification and physiochemical properties of a recombinant bovine growth hormone produced by cultured murine fibroblasts.
Mouse fibroblast cell lines which secrete bovine (b) GH have been generated. This was accomplished by cotransforming mouse L cells (thymidine kinase-negative [TK-] and adenine phosphoribosyl transferase-negative [APRT-]) with DNA molecules encoding the Rous sarcoma virus-long-terminal repeat and bGH genes along with the herpes viral TK gene and the hamster APRT gene. One stable cell line, Pd lambda-pbGH 4-13, was found to secrete approximately 75 micrograms bGH per 24 h/5.0 X 10(6) cells. Media from this cell line were collected for purification of recombinant bGH (rbGH). Purification involved (NH4)2SO4 fractionation, ion-exchange chromatography, and gel filtration on Sephacryl S-200. The rbGH was characterized by bioassay, RIA, radioreceptor assay, and sodium dodecyl sulfate gel electrophoresis. Results of these analyses were compared with those obtained with a highly purified pituitary bGH. In the rat tibia bioassay, rbGH was found to be as potent as pituitary bGH. Results from the RIA, radioreceptor assay, and sodium dodecyl sulfate gel electrophoresis and Western blot analysis also suggested that the rbGH was similar to that of pituitary origin. Amino acid composition, partial (amino-terminal) sequence, and tryptic peptide maps were also found to be similar between the rbGH and pituitary bGH preparations. The amino terminus of the rbGH showed similar heterogeneity to that of the bGH of pituitary origin. We conclude that rbGH which was synthesized, processed, and secreted from transformed mouse fibroblasts possessed almost exactly the same physiochemical properties as pituitary bGH.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001681:Biological Assay; D001846:Bone Development; D002417:Cattle; D002460:Cell Line; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D005347:Fibroblasts / Q000378:metabolism*; D013006:Growth Hormone / Q000096:biosynthesis* / Q000302:isolation & purification; D051379:Mice; D010446:Peptide Fragments; D011863:Radioimmunoassay; D011869:Radioligand Assay; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis*
D000596:Amino Acids; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013006:Growth Hormone