Purification,Action Mechanism In Vitro And Heterologous Expression Of Antitumor Active Protein From Antrodia Cinnamomea Mycelium

Cancer is one of leading causes of death all over the world,and the amounts of new cases and deaths remain high every year,seriously endangering people’s lives and health.Once some malignant tumors are discovered,most of them are at an advanced stage with poor prognosis and low survival rate,thus it’s extremely difficult to treat.In January this year,the State Drug Administration approved the launch of the first original Chinese medicine drug,icariin capsules(acoladine).As a rare medicinal fungus in Taiwan,Antrodia cinnamomea has been proven to have obvious therapeutic effects,such as anti-tumor and anti-inflammatory.Previous studies focused on polysaccharides,triterpenoides,and ubiquinones,but there are relatively few studies on antitumor proteins.In this study,based on the optimization of liquid fermentation of A.cinnamomea in our previousresearch,an A.cinnamomea active protein(ACAP)was isolated and purified from the mycelia.Its antitumor action mechanism was preliminarily explored,and the recombinant expression in Pichia pastoris wasperfomed subsequently.The main findings are as follows:1.The extraction process of A.cinnamomea proteins from mycelia was optimized by single factor experiment and orthogonal design,and the protein concentration was detected by Bradford method.The optimal extraction conditions were determined as: Tris-HCl was used as the extraction buffer at 4℃,and the ratio of material to liquid was 1:20.After 8 h of extraction,the crude proteins were obtained,and the extraction yield was 4.3%.2.After precipitation by ammonium sulfate salting out method,the active protein was purified by anion exchange chromatography and gel filtration chromatography.According to screening the antitumor activity of the eluted fractions,the active protein ACAP was identified.The single protein band in SDS-PAGE gel was analyzed by mass spectrometry,and the target gene sequence was determined through the alignment of its amino acid sequence with the A.cinnamomea transcriptome data.That’s beneficial for the following protein recombinant expression.3.The inhibitory effect of ACAP on tumor cells(HeLa,Hep G2 and Hepa 1-6)was studied by in vitro experiment,and the result showed that ACAP inhibited proliferation of the tumor cells significantly at a lower concentration.The half-inhibitory concentration(IC50)values were 13.10,10.70,and 18.69 μg/mL,respectively.Hep G2 cell apoptosis was detected by flow cytometry,and the antitumor mechanism of ACAP was further explored.Compared to the control group,ACAP mainly induced the late apoptosis of Hep G2 cells,and the proportion of early apoptosis was lower.Western blot analysis of related proteins involved in apoptosis pathway was conducted,and the result revealed that expression level of p53 was up-regulated significantly,while the expression level of caspase-3 was down-regulated significantly.4.After data contrasting,the ACAP-1 gene sequence with the highest coverage with the transcriptome data of A.cinnamomea was determined.The bioinformatics analysis showed that the length of its CDS sequence ACAP-1 is 1080 bp,the molecular weight is about 50 kDa,and the theoretical isoelectric point(pI)is 5.9.Using Primers designed according to the target gene sequence,the target gene fragment was cloneed.And the recombinant plasmid pPIC9K-ACAP was constructed and transferred into Pichia pastoris GS115 strain.The recombinant strains were screened using gradient concentrations of geneticin.Expression of the target recombinant protein was induced using methanol,and its antitumor activity was preliminarily confirmed.