High Expression of the Human Antibacterial Peptide LL-37 in Pichia pastoris and the Detection of Its Activity

Abstract:

Based on the gene sequences encoding human antibacterial peptide LL-37 as registered in Gen- Bank (serial number in GenBank is CAA86115), using the preferential condon of P. pastoris, the antibacterial peptide LL-37 gene 141 bp in length was designed and synthesized. Especially a Kex2 signal cleavage site was fused in 5′end of the antibacterial peptide gene. The modified antibacterial peptide gene was clonedinto the pPICZa-A vector to construct the recombinant expression vector pPICZa-A-LL-37. The SacⅠlinearized plasmid pPICZa-A-LL-37 was transformed into P. pastoris X-33 by electroporation. The transformants were identified by PCR using R2 and 5’ AOX1 specific primers. The concentration of the secreted LL-37 was 206 mg/L. The new peptide, which has a weight of 4.5 kD, could remain its inhibition activity after being treated for more than 3 hours in boiled water. Agrose diffusion assay showed that LL-37 had broad-spectrum antibacterial abilities not only to Gram-negative bacteria but also to Gram-positive bacteria, the MIC to Staphylococcus aureus (CowanⅠ), E. coli K99 and Salmonella pullorum were 1.56 mg/mL, 3.12 mg/mL and 1.56 mg/mL, respectively.