Expression Of Recombinant Human Lysozyme In Bioreactors

Lysozyme (LYZ) is a ubiquitous enzyme in the nature. It can kill bacterium by lysing the bacterial cell wall. Lysozymes are classified into six types according to their molecular structures, molecular weights and sources: plant type lysozyme, bacteria type lysozyme, phage lysozyme, chicken egg-white lysozyme (c-type, clyz), goose egg-white lysozyme (g-tpye, glyz) and invertebrate type lysozyme (i-type). The most common ones in the nature are c-type, g-type and phage lysozymes. Lysozyme is an important defensive factor presented in the humor body liquid and tissues of human and other animals and plays a significant role in the non-specific immunity. It also has many other pharmacological functions, such as anti-bacterium, anti-inflammation, promoting organism recovery, so it is widely used in food and feed industries, scientific research and medical clinical treatments.Human lysozyme (hLYZ) is a 130-animo acid c-type lysozyme with 4 disulfide bonds, and its molecular weight is 14.4kD. Comparing with other kinds of lysozyme, hLYZ has a much different primary structure, but has a very similar tertiary structure. Comparing with chicken egg-white lysozyme which is widely used in clinic, hLYZ has many advantages: it is a natural protein in human body, so it is safe and harmless when used as a drug; its bioactivity to kill bacterium is three times higher than chicken egg-white lysozyme, and its thermal stability is much higher, too; it also has many other specific important functions which are not related with its catalysis, such as anti-virus and anti-tumor and improving immunization. With the in-depth understanding of hLYZ functions and the increasing demand in the market, more and more attention is paid in the large-scale production and application of hLYZ.Currently, most hLYZ is extracted from human milk and placenta. Limited by the source and scale, the problems for production of hLYZ are low yield, unstable quality and high cost. Utilizing bioreactor to produce hLYZ can make positive contribution to solve these problems, and can also provide an alternative way to hLYZ.The present study tried to use bioreactors to express recombinant hLYZ in three different expression systems: Escherichia coli, Pichia pastoris and plant oil body. The major achievements in this study were as follows:â‘ two different hLYZ genes were designed and synthesized according to the plant and E.coli codon bias usages, respectively, and in both genes a his-tag and a enterokinase cleavage site were introduced to simplify the purification procedures later on.â‘¡an E. coli expression vector pET32a-hLYZe was successfully constructed, which was subsequently transferred into E. coli strain BL21. After induction by IPTG, the engineered strain expressed recombinant hLYZ with specific immunogenicity, which existed as insoluble inclusion bodies, which was confirmed by SDS-PAGE and Western blot analyses. The recombinant protein was purified by Ni+ affinity chromatography after a series of denaturation, solution and renaturation procedures, and then analyzed for the hLYZ bioactivity by turbidimetric method. The result showed that the recombinant hLYZ expressed by E. coli was bioactive.â‘¢a P. pastoris secreton expression vector pPICZ?-hLYZ was successfully constructed, which was then transferred into P. pastoris strain GS115. After induction by methanol, the engineered strain expressed recombinant hLYZ with specific immunogenicity, which was confirmed by SDS-PAGE and Western blot analyses. The recombinant protein was purified by Ni+ affinity chromatography and measured for hLYZ concentration by Bradford assay, and then analyzed for the hLYZ bioactivity by turbidimetric method. The result indicated that the concentration of expressed hLYZ was 324mg/L, the bioactivity was 4473U/mL, and the specific activity was approximately 13800U/mg, which was higher than conventional hLYZ extracted from human milk.â‘£a"Twin T-DNAs"-containing plant expression vector p2300-OP-(his)-OG-hLYZ was successfully constructed, which was then transferred into Agrobacterium tumefaciens strain GV3101. Twenty-seven kanamycin-resistant Arabidopsis plant were regenerated by Agrobacterium-mediated transformation.