Expression Of The Recombinant Human Lysozyme In Pichia Pastoris And Its Application In Screening Amyloid Fibrosis Inhibitors

Lysozyme is a kind of non-specific immune factors,compatibility with the human body has a natural,no side effect to human body,and has antibacterial,antiviral and anticancer etc,in clinical,food industry and animal husbandry in areas such as widely used,but it is still not implemented restructuring lysozyme high-yield strains and large-scale production technology breakthrough.In addition,human lysozyme is an ideal model for drug screening of inhibitors through amyloid fibrosis in the pathogenesis and drug research of Hellzheimer’s disease,because it is a protein containing a variety of typical secondary structures and disulfide bonds.Based on this,this study focused on the construction,expression and fermentation process of recombinant human lysozyme Pichia pastoris with high yield,as well as the application of human lysozyme in the screening of amyloid fibrosis inhibitors.The following are the main contents of the research work in two aspects:1.Construction of recombinant human lysozyme pichia pastoris expressing strain and study on fermentation technology.In this study,the expression plasmid p PIC9K containing human lysozyme gene was integrated into Pichia pastoris by the method of electric transformation,and the engineered strain of human lysozyme Pichia pastoris was constructed.The strain was identified as MUTS type by phenotypic identification.A recombinant strain of human lysozyme Pichia pastoris with high copy was further identified by G418 resistance screening.At the same time,a high performance liquid chromatography method for detecting human lysozyme activity was established,and the methodology was verified.This method can completely replace the biological method for detecting lysozyme activity,and improve the efficiency and accuracy of analysis and detection.In addition,the single-factor experiment was carried out on the fermentation conditions of Pichia pastoris strains with high copy of human lysozyme at the shaking flask level.By investigating the influence of different induction time,temperature,methanol content,p H value and liquid volume on the content of human lysozyme induced expression,the optimal fermentation conditions of recombinant human lysozyme Pichia pastoris strain were optimized as follows:A 25 m L flask（500 m L）with a p H of 5.5 was placed in a shaker at 28℃and a rotating speed of 220 rpm for 120 h.During the induction process,the induced methanol content was kept at 2%and was supplemented every 24 h.Then it was transferred to a fermenter to optimize the methanol feeding method.The effects of four methanol feeding methods:constant rate feeding,p H feedback feeding,DO feedback feeding and exponential feeding,on the induced expression of human lysozyme were investigated using the bacterial body mass（dry weight,wet weight）,enzyme activity and SDS-PAGE as evaluation indexes.The optimized results showed that the highest expression of human lysozyme reached 49422.78 U/mg after 132 h induction in the fermentation tank at 28℃and p H5.5 under the feeding mode of DO feedback flow.The optimized results provided a new idea for the development of industrial induction of human lysozyme expression in Pichia pastoris.2.Application of human lysozyme in screening amyloid fibrosis and its inhibitorsAmyloid proteins were recognized as the crucial cause of many senile diseases including Alzheimer’s disease（AD）,Parkinson’s disease（PD）,and type 2 Diabetes Mellitus（T2DM）,and the establishment of amyloid fibrosis models in vitro is considered to be a common strategy for finding inhibitors of amyloid fibrosis.Previous researches have demonstrated that natural products from plants can maintain the native conformations of proteins and exhibit the therapeutic activities for neurodegenerative diseases.In this study,the inhibitory effects of Sennoside A（SA）and Sennoside C（SC）on the amyloid fibrillation were evaluated by the combination of biophysical approaches and molecular docking tool using human lysozyme（HL）as an amyloid-forming model.The results of thioflavin-T（Th T）,8-anilino-1-naphthalenesulfonic acid（ANS）and congo red（CR）assays indicated that both SA and SC could inhibit the amyloid fibrillation of HL in a dose-dependent manner.The IC₅₀ value of SA and SC on HL fibrillation was 200.09μM and 186.20μM,respectively.These findings were further verified by transmission electron microscopy（TEM）and atomic force microscopy（AFM）,which showed that the addition of SA or SC could sharply reduce the amyloid fibrillation of HL.Additionally,the interactions of HL with SA and SC were investigated by the steady-state fluorescence spectra and molecular docking studies.The results suggested that both SA and SC could bind to the binding pocket of HL and form a stable complex mainly via hydrogen bonds,van-der-Waals forces and hydrophobic interactions.In conclusion,our experiments revealed that both SA and SC can significantly inhibit the amyloid fibrillation of HL.