Characterization of human type III collagen expressed in a baculovirus system. Production of a protein with a stable triple helix requires coexpression with the two types of recombinant prolyl 4-hydroxylase subunit.

An efficient expression system for recombinant collagens would have numerous scientific and practical applications. Nevertheless, most recombinant systems are not suitable for this purpose, as they do not have sufficient amounts of prolyl 4-hydroxylase activity. Pro-alpha 1 chains of human type III collagen expressed in insect cells by a baculovirus vector are reported here to contain significant amounts of 4-hydroxyproline and to form triple-helical molecules, although the Tm of the triple helices was only about 32-34 degrees C. Coexpression of the pro-alpha1(III) chains with the alpha and beta subunits of human prolyl 4-hydroxylase increased the Tm to about 40 degrees C, provided that ascorbate was added to the culture medium. The level of expression of type III procollagen was also increased in the presence of the recombinant prolyl 4-hydroxylase, and the pro-alpha 1(III) chains and alpha1(III) chains were found to be present in disulfide-bonded molecules. Most of the triple-helical collagen produced was retained within the insect cells and could be extracted from the cell pellet. The highest expression levels were obtained in High Five cells, which produced up to about 80 microg of cellular type III collagen (120 microg of procollagen) per 5 X 10(6) cells in monolayer culture and up to 40 mg/liter of cellular type III collagen (60 mg/liter procollagen) in suspension. The 4-hydroxyproline content and Tm of the purified recombinant type III collagen were very similar to those of the nonrecombinant protein, but the hydroxylysine content was slightly lower, being about 3 residues/1000 in the former and 5/1000 in the latter.