Study On The Expression Of Recombinant Human Lysozyme Fusion Gene In Pichia Pastoris

Human lysozyme (hLY) is an important defensive factor presented in the humor body liquid and tissues of human. It has many pharmacological functions, such as anti-bacterium, anti-inflammation, promoting organism recovery and enhancing immunity. The biological origin and own characteristics of human lysozyme, which show great advantages in low toxicity and anti-drug resistance, provide a new choice for clinical medication.Since human lysozyme is drawn from milk and placenta in only small quantities, furthermore, the cost of its separation and purification is high, it's hard to be produced on a large scale. Hence, widespread application of human lysozyme should be depended on biotechnological means. Up till now, heterologous human lysozyme has been expressed in various organisms. Among them, Escherichia coli and Pichia pastoris expression systems have been used mostly in researches.In order to balance human lysozyme's positive charge and lower its toxity to host cells, we respectively fused human lysozyme with anionic peptide (AP) and xylanase (Xyn). Between the two genes, an enterokinase cleavage site or a flexible linker peptide was introduced. Then the fusion proteins expressed were secreted into medium in the form of inactivity or week activity. The fusion proteins, after enterokinase cleavaging, released human lysozyme and xylanase with bioactivity.The major achievements in this study were as follows:â‘ A partial sequence propiece was selected to be anionic peptide and synthesized according to the P. pastoris codon bias usages, and in both genes a His-tag and an enterokinase cleavage site were introduced to simplify the purification procedures later on. The eukaryotic expression vector carrying AP-hLY fusion gene was constructed and secreting expressed in P. Pastrois. However, protein electrophoresis analysis result showed that the molecular weight of recombinant protein was 38.1 kD, much higher than that of theoretical.â‘¡hLY gene and Xyn gene were linked with specific primers. The linker sequence between hLY gene and Xyn gene contained a site which recognized by enterokinase or a flexible linker peptide. Subsequently, both hLY-EKsite-Xyn and hLY-Linker-Xyn were successfully constructed and expressed in P. Pastrois. The recombinant proteins were expressed by protein electrophoresis detection. Determination of the supernatant activity by using improved Shugar method and DNS method, elucidated that the lysozyme activity of hLY-Linker-Xyn was 180 U/mL, little higher than that of hLY-EKsite-Xyn 170 U/mL. But the xylanase activity was respectively 158 U/mL and 185 U/mL, in contrast. Then the fusion proteins were extracted and purified by means of ultrafiltration concentration and Sephadex G-75 gel chromatography.â‘¢After enterokinase cleavage, the activity analysis of the recovered hLY-EKsite-Xyn indicated that lysozyme activity of released proteins was 520 U/mL, and xylanase activity was 244 U/mL.The both bioactivities had considerable improvement as we anticipated.