Recombinant expression of Bacillus cereus collagenase colR75E in Pichia pastoris

Abstract:

[Objective] In order to find a safe and stable method to produce collagenase in vitro, we expressed the Bacillus cereus collagenase colR75E in Pichia pastoris. [Methods] With the Bacillus cereus genomic DNA as template, we successfully amplified the collagenase colR75E DNA fragment by PCR and cloned it into pPICZαA plasmid. The pPICZαA/colR75E recombinant plasmid was lineared with Sac I, and then the lineared plasmid was transformed into Pichia pastoris X-33 competent cell in order to integrate the inducible AOX1 promoter controlled colR75E fragment into Pichia pastoris X-33 genomic DNA. The successfully integrated Pichia pastoris X-33 strains was cultured and induced by methanol addition. To acquire the highest production, the optimized conditions for ColR75E collagenase expression in Pichia pastoris X-33 were investigated here. After induction, we purified recombinant ColR75E collagenase in supernatant sequentially by ammonium sulfate precipitation, desalting and affinity capture. Finally, the recombinant collagenase ColR75E was analyzed by catalytic activity assay, SDS-PAGE, zymography, type I collagen proteolysis and substrate specificity assay. [Results] The highest level of collagenase ColR75E induction was gained under pH 6.0 for 72 hours incubation by 2.5% methanol. As expected, the molecular weight of the recombinant collagenase is nearly 110 kD to ColR75E. The results of zymography and type I collagen degradation analysis uncovered that the recombinant collagenase ColR75E had an excellent collagen proteolysis activity. Its specific activity after purification reached to nearly 4.977 U/mg under standard conditions. The recombinant collagenase ColR75E exhibited specific proteolysis to type I collagen, but not to BSA, Casein or Lysozyme. [Conclusion] Pichia eukaryotic expression system is suitable for the expression of Bacillus cereus collagenase ColR75E, which supplied a good basement both for its subsequent theoretic research and industrial exploitation.

Key words:Bacillus cereus, Collagenase, Pichia pastoris, Type I collagen