Intracellular Expression, Isolation and Purification of LfcinB and EGFP Fusion Protein in Pichia pastoris
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Abstract:
In this study, the enhanced green fluorescent protein (EGFP) gene fragment was ligated with the N-terminus of the antimicrobial peptide bovine lactoferrin peptide (LfcinB-His) gene fragment to construct an intracellular expression vector with EGFP as the reporter gene. The recombinant protein EGFP-LfcinB-His was fused and expressed, and its activity was determined. The recombinant expression plasmid was electroporated into Pichia pastoris GS115, and the positive transformants were screened. The fermentation expression was induced by methanol, and strong fluorescence was detected under the excitation light of 488 nm. When the yeast cell culture reached the highest density, the cells were collected, the cell walls were broken, and the cells were lysed with a yeast cell protein lysate. The target protein (LfcinB-His) band with a relative molecular mass of 4.1 ku was detected by Tricine-SDS-PAGE, after the lysate was purified by steps such as ultrafiltration concentration, Ni-NTA affinity chromatography and formic acid lysis. Finally, a LfcinB-His ultrafiltration concentrate with a purity of 90.32% was obtained through the determination and analysis by liquid chromatography-mass spectrometry, which had different degrees of inhibition on the six strains of pathogenic bacteria tested, and the inhibitory concentration range was 16~64 μg/mL. In summary, this study provides a good methodological reference for the expression of small molecular peptides in Pichia pastoris, and also lays a foundation for further research on the biological activity and high-density fermentation of LfcinB.
