A type I collagen reporter gene construct for protein engineering studies

A type I collagen reporter gene construct, designed to facilitate detailed analysis of the consequences of introduced structural and regulatory mutations on collagen biosynthesis and participation in the extracellular matrix, was produced by sitedirected mutagenesis of the mouse COLIAI gene. The reporter construct, pWTCI-Ile822, carried a single base change which converted the codon for amino acid 822 of the triple helix from methionine to isoleucine. This change allowed the reporter protein, [Ile822]al(I), to be distinguished from the wild-type al(I), and quantified, by its altered CNBr cleavage pattern. In mouse Movl3 cells, which synthesize no endogenous proal(I), reporter chains associated with endogenous proa2(I), formed pepsinstable triple helices and were secreted efficiently from the cell.