Heterologous Expression and Functional Characterization of CAP۱۸ from Oryctolagus cuniculus

Background: Antimicrobial peptides belong to the innate defence system of creatures. These peptides attach to the bacterial membrane in order to die microorganisms by penetrating them. Hence, biotechnology researchers pay more attention to produce antimicrobial peptides for use in various fields. The studies showed that rabbit tissue with inflammation and skin ulcers would be producing CAP۱۸ peptide, which belongs to the cathelicidin group. Methods: In this study, the optimized sequence of the cap۱۸ gene was placed into the pPICZAα plasmid after the alpha-factor signal and transformed into Pichia pastoris (X-۳۳ strain). Purification of the recombinant peptide was done based on its histidine tail at C-terminal, and western blotting method was used to demonstrate the purification of rCAP۱۸. The antibacterial activity of the purified and desalted rCAP۱۸ was investigated at different concentrations against pathogenic bacteria. Results: The maximum expression level of rCAP۱۸ (۱۷.۵ kDa) was seen ۹۰ h after induction of alcohol oxidase I (AOX۱) promoter with methanol. The concentration of rCAP۱۸ was ۳۳ mg/L after purification with Ni-NTA Sepharose column. The function of rCAP۱۸ (۴.۳, ۵.۷, ۷ μg/ml) was investigated against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Results showed that %CFU/cm۲ reached ۲۸% after P. aeruginosa cells treatment with ۷ μg/ml of rCAP۱۸. Conclusions: This study presented the findings related to heterologous expression of cap۱۸ gene, and evaluation of rCAP۱۸ antibacterial effects. Our results showed that rCAP۱۸ plays a significant role in inhibiting bacterial growth, especially Gram-negative bacteria.

Keywords:

Antibacterial activity , Cathelicidin , Recombinant CAP۱۸ , Heterologous expression.