BIOSYNTHESIS OF RECOMBINANT HUMAN PRO-ALPHA-1(III) CHAINS IN A BACULOVIRUS EXPRESSION SYSTEM - PRODUCTION OF DISULFIDE-BONDED AND NON-DISULFIDE-BONDED SPECIES CONTAINING FULL-LENGTH TRIPLE HELICES

We have investigated the expression of human procollagen III by insect cells infected with a recombinant baculovirus carrying cDNA for the p ro-alpha 1(III) chain of type-III collagen. A high level of expression was obtained, and a small proportion of the heterologously expressed pro-alpha 1(III) chains formed normally disulphide-bonded procollagen III, which was secreted into the culture medium. This species displaye d a melting temperature (T-m) of approx. 38 degrees C as assessed by i ts resistance to digestion by a mixture of trypsin and chymotrypsin, s lightly lower than that of 39.5 degrees C for procollagen III synthesi zed by cultured human dermal fibroblasts, and reflected a slight degre e of under-hydroxylation of prolyl residues. This is possibly a conseq uence of the lower incubation temperature of insect cells, or of an in sufficiency of prolyl hydroxylase activity within them. A significant proportion of the expressed chains formed trimeric molecules of simila r thermal stability containing an apparently full-length triple-helica l region, but were not disulphide-bonded and not secreted. In addition to providing a source of recombinant human procollagen III, the syste m promises to be useful in the study of procollagen chain association and subsequent folding.