title: Biosynthesis of recombinant human pro-alpha 1(III) chains in a baculovirus expression system: production of disulphide-bonded and non-disulphide-bonded species containing full-length triple helices.
abstract: We have investigated the expression of human procollagen III by insect cells infected with a recombinant baculovirus carrying cDNA for the pro-alpha1(III) chain of type-III collagen. A high level of expression was obtained, and a small proportion of the heterologously expressed pro-alpha1(III) chains formed normally disulphide-bonded procollagen III, which was secreted into the culture medium. This species displayed a melting temperature (Tm) of approx. 38 degrees C as assessed by its resistance to digestion by a mixture of trypsin and chymotrypsin, slightly lower than that of 39.5 degrees C for procollagen III synthesized by cultured human dermal fibroblasts, and reflected a slight degree of under-hydroxylation of prolyl residues. This is possibly a consequence of the lower incubation temperature of insect cells, or of an insufficiency of prolyl hydroxylase activity within them. A significant proportion of the expressed chains formed trimeric molecules of similar thermal stability containing an apparently full-length triple-helical region, but were not disulphide-bonded and not secreted. In addition to providing a source of recombinant human procollagen III, the system promises to be useful in the study of procollagen chain association and subsequent folding.
authors.affiliation: Institute of Biomedical Science, Terumo Corporation R. & D. Centre, Kanagawa, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Tomita
authors.forename: N
authors.initials: N
authors.lastname: Ohkura
authors.forename: M
authors.initials: M
authors.lastname: Ito
authors.forename: T
authors.initials: T
authors.lastname: Kato
authors.forename: P M
authors.initials: PM
authors.lastname: Royce
authors.forename: T
authors.initials: T
authors.lastname: Kitajima
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D002918:Chymotrypsin / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry*; D004355:Drug Stability; D015870:Gene Expression; D006358:Hot Temperature; D006801:Humans; D008969:Molecular Sequence Data; D011347:Procollagen / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis; D018411:Spodoptera / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
chemical_list: D000596:Amino Acids; D004220:Disulfides; D011347:Procollagen; D011994:Recombinant Proteins; D002918:Chymotrypsin; D014357:Trypsin; D001205:Ascorbic Acid