Infectious diseases caused by antibiotic multidrug-resistant microorganisms are major causes of morbidity and mortality in humans. Hence, there is an urgent need to search for new antimicrobial agents. Initially known as a defensive effector in the innate immunity of certain organs of the human body, human goose-type lysozyme 2 (hLysG2) has been shown to possess therapeutically useful potential against multidrug-resistant microorganisms. Developing a novel strategy for large-scale production that provides high yields of this protein with high purity, quality, and potency is critical for pharmaceutical applications. To overcome the issues related to prokaryotic expression, here we report the production of recombinant hLysG2 (rhLysG2) using the methylotrophic yeast Pichia pastoris as expression host. The strong inducible alcoholoxidase 1 (AOX1) promoter was used to drive expression of the optimized hLysG2 gene. Under the optimal expression conditions, the lyric activity of rhLysG2 reached 113 U/mL of culture supernatant in shake flask cultivation and this was increased to 2084 U/mL in fed batch fermentation. Using chitin affinity chromatography and size-exclusion chromatography, rhLysG2 was produced with a yield of 137 mg/L, purity of > 99%, molecular weight of 21,504.6 Da, and specific activity of 13,500 U/mg. In vitro assays indicated that rhLysG2 possessed muramidase activity, isopeptidase activity, and free radical scavenging activity. This report describes an efficient strategy for the production of biologically active rhLysG2 in P. pastoris on a large scale with a high yield, which provides a solid foundation for possible future pharmaceutical applications.
[1]Shanghai Univ Med & Hlth Sci, Sch Clin Med, 279 Zhouzhu Rd, Shanghai 201318, Peoples R China.
[2]Fudan Univ, Sch Life Sci, 2005 Songhu Rd, Shanghai 200438, Peoples R China.
[3]Shanghai Univ Med & Hlth Sci, Sch Basic Med, 279 Zhouzhu Rd, Shanghai 201318, Peoples R China.
(8.0+极速模式)