Multiple Copies of the Fusion Gene cflyC-mzfDB3 Enhance the Expression of a Hybrid Antimicrobial Peptide in Pichia pastoris

The codon-optimized zebrafish beta-defensin 3 mature peptide gene mzfDB3 and channel catfish c-type lysozyme gene cflyC were used to design the fusion gene cflyC-mzfDB3. In the fusion protein cflyC-mzfDB3, 4xGly flexible amino acid linker with an enterokinase cleavage site DDDDK was designed to link the mzfDB3 to the C-terminus of cflyC, which potentially contributes to digestion of the recombinant cflyC-mzfDB3 by endogenous enterokinases. The fusion protein was expressed in Pichia pastoris X-33 using expression vectors harboring 1-, 2-, or 4-copies, respectively, of the expression cassette. The cflyC-mzfDB3 gene copy numbers of P. pastoris transformants were quantified using real-time quantitative PCR. Their expression yields showed that the expression level in the 4-copy cflyC-mzfDB3 transformant was 2.67-fold higher than that in the 1-copy cflyC-mzfDB3 transformant, demonstrating that the increase in the cflyC-mzfDB3 gene copy number resulted in increased protein expression. The culture medium supernatant containing recombinant cflyC-mzfDB3 exhibited antibacterial activity against Gram-positive Listeria monocytogenes and Gram-negative Pseudomonas aeruginosa, indicating that there may be a synergistic effect of mzfDB3 and cflyC on the peptide's antibacterial activity. In addition, cell-free P. pastoris medium may be a potential candidate for further development as a natural hybrid antimicrobial solution.