Fusion Expression,Purification And Identification Of Human Islet Amyloid Polypeptide

The aggregation of amyloid into insoluble starch fibers can lead to various diseases such as Alzheimer's disease,Parkinson's disease and type 2 diabetes(T2DM).Amyloid deposits formed by aggregation of human amylin(hIAPP)in the pancreas of T2DM patients are its main pathological features.Aggregation of hIAPP monomers to form aggregates rich in ?-sheet structures leads to islet ? cell dysfunction and death and ultimately T2DM.Therefore,how to suppress hIAPP aggregation has become a research hotspot to overcome T2DM.At present,hIAPP used in in vitro experiments is mostly prepared by solid phase chemical synthesis(SPPS),but the residual amino acids and short peptides in the synthesis process seriously affect the aggregation characteristics and toxicity of hIAPP.Based on the amino acid sequence of hIAPP,firstly,this study added protein Linker and Factor Xa/TEV protease cleavage site between the target protein and the fusion tag(GST and MBP).The hIAPP gene was then preferentially synthesized according to the E.coli codon and cloned into different expression vectors.Recombinant plasmids pET22b-His-GST-hIAPP and pMAL-hIAPP containing target gene were obtained by transformation.The recombinant plasmid was verified by double digestion and gene sequencing.The correct recombinant plasmid was transformed into the expression host E.coli BL21(DE3).The plasmid was extracted using a kit and PCR was verified using the extracted plasmid as a template,and that the correct engineered bacteria were used for late induction fermentation.In order to increase the expression level of the fusion protein,the effects of temperature and induction time on the expression of the target protein were systematically investigated.It was found that the cell concentration of OD600 was 0.4,and the induction of 32 h at 12? was the best induction condition for the engineered bacteria.A single colony of the constructed engineered bacteria was inoculated into a 5 mL LB tube(the final concentration of ampicillin resistance was 50 ?g/mL).After 12 h of culture,1 mL(2%inoculum)was transferred to 50 mL of LB medium(the final concentration of ampicillin was 50 ?g/mL).Culture at 37 ? until the concentration of bacteria reaches OD600 of 0.4-0.8 The inducer IPTG was added to a final concentration of 0.5 mmol/L,and the fermentation was induced by shaking at 12? and 120 r/min for 30-34 h.After induced fermentation,the cells were collected by centrifugation,sonicated at low temperature,and the fusion protein was purified by affinity chromatography on a nickel column.The fusion protein MBP-hIAPP was cleaved using TEV protease.Firstly,the effect of enzyme digestion conditions on enzyme digestion was studied.It was found that the reaction at 16? for 6 h was the best enzyme digestion condition.The hIAPP was further purified by size exclusion chromatography.The isolated hIAPP monomer was then electrophoretically analyzed.The molecular weight was verified by MALDI-TOF mass spectrometry to be 3931.57,which is consistent with the theoretical molecular weight of hIAPP.Finally,the concentration of hIAPP was determined by Nanodrop and the yield of hIAPP was calculated to be about 4-6 mg/L.After biosynthesis to obtain hIAPP monomer,it is used to study its aggregation characteristics and cytotoxicity in vitro,and provide a targeted drugs for the treatment of type 2 diabetes.