Production of recombinant human type I procollagen trimers using a four-gene expression system in the yeast Saccharomyces cerevisiae

The expression of stable recombinant human collagen requires an expression system capable of post-translational modifications and assembly of the proc ollagen polypeptides. Two genes were expressed in the yeast Saccharomyces c erevisiae to produce both propeptide chains that constitute human type I pr ocollagen. Two additional genes were expressed coding for the subunits of p rolyl hydroxylase, an enzyme that post-translationally modifies procollagen and that confers heat (thermal) stability to the triple helical conformati on of the collagen molecule. Type I procollagen was produced as a stable he terotrimeric 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 heterotr imers in Saccharomyces, Expression of these four genes was sufficient for c orrect assembly and processing of type I procollagen in a eucaryotic system that does not produce collagen.