Methods for production of recombinant enzymes for collagen degradation

Abstract: In this thesis eight enzymes have been identified with documented ability to degrade collagen or its hydrolyzed products. Collagen can be found in skin, bones, and connective tissue. Hydrolyzed collagen peptides are utilized in food, disease treatment, skincare and training supplements. The studied enzymes are: Clostridium histolyticum collagenases ColG and ColH, Grimontia hollisae collagenase, Bacillus cereus collagenase ColA, subtilisin, trypsin, pepsin and papain. Two organisms were used to develop cloning strategies, Escherichia coli and Pichia pastoris. For E. coli the enzymes have been optimized for cytoplasmic expression with pET22b+ expression vector, for periplasmic expression with pET22b+ in frame with pelB signal peptide and for extracellular expression with pAES40 vector with YebF protein. Extracellular expression of the enzymes with P. pastoris was optimized for pPICZα expression vector with α-factor signal sequence. Two cloning strategies have been developed for the four expression systems which included subcloning, protein production and purification. Furthermore, possible methods for evaluating the produced enzymes were investigated. Additionally, a strategy to determine the most effective enzyme combination for breakdown of collagen was developed which resulted in a maximum of 16 tests. The optimized genes were compared between the species and a difference in codon bias was found as well as between different algorithms which showed that different tools gave diverse sequences. The cloning strategy developed for E. coli was experimentally tested with G. hollisae collagenase and subtilisin Carlsberg. The cloning was not successful as no growth of transformed cells were observed. It was discovered that the size of vector backbone and genes of interest caused difficulties for purification of genes, but it was postulated that with additional restriction enzymes or PCR and different competent cells the enzymes could have been successfully expressed.