High expression of bovine lactoferrin N-lobe in Pichia pastoris and its antibacterial activity

Abstract:

[Objective] We cloned the bovine lactoferrin N-lobe (BLF-N) into the genome of Pichia pastoris and enabled the heterologous expression of BLF-N through the optimization of its gene codon and fermentation conditions.Furthermore,we studied the antibacterial activity of the recombinant protein.[Methods] With BLF gene as template,BLF-N gene was optimized according to the codon bias of P.pastoris.On this basis,the recombinant expression vector pPIC9K-UBLF-N was constructed and transformed into P.pastoris GS115 by electroporation to yield the recombinant P. pastoris GS115/pPIC9K-UBLF-N.Through high-copy screening and optimization of fermentation conditions,BLF-N expression was improved and then evaluated by SDS-PAGE.The inhibition of the recombinant BLF-N on Gram-positive and Gram-negative bacteria was explored.[Results] SDS-PAGE results showed the efficient soluble expression of BLF-N with molecular weight of about 37 kDa (consistent with the theoretical molecular weight) in P. pastoris GS115.A transformant resistant to 3 mg/mL genomycin G418 was obtained through high-copy screening.The highest recombinant BLF-N yield (50.5 mg/L) was achieved under the following optimized fermentation conditions:0.2%(V/V) methanol,30℃,and pH 5.0.Recombinant BLF-N demonstrated strong inhibition on Escherichia coli and Staphylococcus aureus and higher antibacterial activity than commercial BLF.[Conclusion] Through high-copy screening and optimization of fermentation conditions,we achieved the efficient expression of BLF-N in P. pastoris and the recombinant BLF-N presented significantly higher antibacterial activity than the commercial BLF.This study lays a foundation for the efficient and green preparation and industrial production of BLF-N and provides theoretical guidance for research on the difference in structure-function relationship between BLF and BLF-N.