Construction of Recombinant Human Collagen III Variants and Genomic Manipulations in Yeast for Collagen Yield Optimization
As the most abundant protein in the extracellular matrix, collagen is an attractive tissue engineering scaffold. IKVAV, a peptide sequence native to the protein laminin, is widely known for its roles in neuroregeneration. Using a computational algorithm, IKVAV-containing collagen variants were successfully assembled in a de novo fashion. The incorporation of IKVAV into the collagen sequence required the interruption of the characteristic Gly-X-Y repeat. These variants, however, were able to retain their triple-helical form, and the one examined yielded a melting temperature comparable to that of our baseline recombinant collagen. Accessibility of IKVAV within the collagen structure was not confirmed, and cell adhesion assays indicate a lack of cell binding to the variants tested. Further studies are necessary to draw definite conclusions.
In our system, the collagen gene is expressed under the GAL1 promoter in Saccharomyces cerevisiae, necessitating cultivation of the yeast in galactose-containing medium for induction. To optimize collagen yield, the REG1 and GAL1 genes were deleted to inhibit catabolite repression and galactose consumption, allowing growth in glucose under constant inducer concentration. Unlike the ΔREG1 strain, the ΔREG1ΔGAL1 strain did not show improved growth in glucose. Upon transformation of a S. cerevisiae CEN/ARS plasmid carrying the collagen gene, the ability of both variants to produce triple-helical collagen was confirmed by circular dichroism. Initial Western blots did not show increased collagen production by the ΔREG1ΔGAL1 strain. However, the strain did allow collagen production during growth on glucose, and the concentration of the galactose inducer can likely be significantly reduced.