Production of Recombinant Human Type I Procollagen Trimers Using a Four-gene Expression System in the Yeast Saccharomyces cerevisiae

Abstract The expression of stable recombinant human collagen requires an expression system capable of post-translational modifications and assembly of the procollagen polypeptides. Two genes were expressed in the yeast Saccharomyces cerevisiae to produce both propeptide chains that constitute human type I procollagen. Two additional genes were expressed coding for the subunits of prolyl hydroxylase, an enzyme that post-translationally modifies procollagen and that confers heat (thermal) stability to the triple helical conformation of the collagen molecule. Type I procollagen was produced as a stable heterotrimeric helix similar to type I procollagen produced in tissue culture. A key requirement for glutamate was identified as a medium supplement to obtain high expression levels of type I procollagen as heat-stable heterotrimers inSaccharomyces. Expression of these four genes was sufficient for correct assembly and processing of type I procollagen in a eucaryotic system that does not produce collagen.