In this study, an episomal Pichia pastoris expression vector was constructed for the recombinant expression of human lysozyme. The commonly used expression vectors in P. pastoris are mainly integrative ones. When the integrative plasmid repeatedly integrates into the chromosome of P. pastoris by homologous recombination, the expression level of heterologous proteins will increase with the increasing copy number of the gene. However, in P. pastoris, stable episomal expression vectors benefit the construction of mutation libraries of heterologous genes and subsequent high-throughput screening. In this paper, the autonomously replicating sequence (pARS) from Kluyveromyces lactis was inserted into the yeast expression vector pPICZαA to obtain the episomal expression vector pPICZαA-pARS. The optimized gene sequence hLYZ was cloned and ligated to the episomal expression vector to construct the recombinant expression vector pPICZαA-hLYZ-pARS, which was then transformed into P. pastoris strain X33 by electroporation. After 72 h of cultivation and induction by methanol in shake-flask, the enzyme activity was 340 U/mL in the supernatant. Then the human lysozyme was purified by nickel affinity chromatography, and the concentration of purified human lysozyme was 0.954 mg/mL determined by BCA. In this study, a P. pastoris engineering strain harboring an episomal vector to express and secrete human lysozyme had been successfully constructed, which laid a foundation for the further studies on using modern mutagenesis technology to screen for human lysozyme mutant with higher antibacterial activity and wider antibacterial spectrum.
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