Prokaryotic Expression And Purification Of Cellulose Of Inonous Obliquus

Inonotus obliquus(Ach.exPers.)Pilat is a very precious and popular fungi.Sclerotium as main medicinal parts with remarkable curative effects on antitumor?resistance to high blood pressure?fall blood sugar and enhance immunity has remarkable curative effect.However,the wild resource of Inonotus obliquus is very scarce,and the wild species on the birch has a good medicinal value after 10-15 years.So the exploration of the artificial cultivation of Inonotus obliquus still is imminent.The cultivation of Inonotus obliquus sclerotium has made successful achievement,but the yield is low.Research shows that the changes of extracellular enzyme activity are closely related with the growth of sclerotium.In this study,JL01 strain was adopted to be test materials.We continue to study the factors of sclerotium yield based on molecular level of cellulose enzyme,in order to further understand the secretion regularity of extracellular enzyme protein function and expression,and provides the theory basis for establishing efficient heterologous expression vector.The specific results are shown blow:1.We obtained the full length ORF sequence of IO-BGL and IO-EG in Inonotus obliquus.A full-length ORF of IO-BGL gene contained 2583bp,which was same as the BGL gene published in Genbank.However,the full length ORF sequence of IO-EG lack a base T compared with Genbank.The new IO-EG gene was named eg I and predicted to encode 382-amino acid protein.Protein molecular weight was 40.1 kDa,and isoelectric point was 4.78.Functional analysis of eg I showed that it belongs to the cellulose enzyme hydrolysis of glycosidic enzyme family 5;2.Prokaryotic expression vector pET-30a-eg I and pET-30a-bgl were successfully constructed;3.Two prokaryotic expression vectors were successfully constructed and then transformed into Trans BL21(DE3).SDS-PAGE respectively proved IO-EG and IO-BGL protein mainly expressed as the form of inclusion body in E.coli,and recombinant protein molecular mass was 40 kDa and 90 kDa,respectively;4.Purification of the target protein was adopted Ni-IDA protein purification kit under the condition of the degeneration of the target protein.IO-EG protein purity was 88%and IO-BGL protein purity was 75%after purification.IO-EG protein concentration was 0.5 mg·mL-1 with a total of around 3.0 mg protein,IO-BGL protein concentration was 0.65 mg·mL-1 with a total of around 3.6 mg protein after concentration.5.Western Blotting analysis of the purified protein showed that IO-EG protein has the single line,however IO-BGL protein has another 35 KDa line.The reason of the mixed line was predicted to be degradation of IO-BGL protein.