Expression, Characterization and Purification of Latcripin-5 from Lentinula edodes Strain C91-3 and its In Vitro Anticancer Activities
Expression, Characterization and Purification of Latcripin-5 from Lentinula edodes Strain C91-3 and its In Vitro Anticancer Activities
Akbar Hussain1, Syed Riaz ud Din1, Kavish Hasnain Khinsar1, Muhammad Noman Ramzan2, Min Huang1, Anhong Ning1, Xiaoli Wang1, Mintao Zhong1* and Bibi Nazia Murtaza3*
ABSTRACT
Lentinula edodes C91-3 is an edible mushroom with demonstrated medicinal activity against various types of cancer in vitro and in vivo. The gene for Latcripin-5 (LP-5) was obtained from Lentinula edodes strain C91-3 and inserted into a pET32a (+) vector, which was then, expressed in the Rosetta gami (DE3) strain. The purified LP-5 protein was analyzed using SDS-PAGE and western blot. The optimal solubilization parameters were found to be 0.6 mM IPGT for 6 h incubation at 37℃. The solubilized protein was refolded using a refolding buffer and then dialyzed and concentrated using a dialysis buffer and PEG 20000, respectively. Phyre2 bioinformatics tool was used for protein modelling. The concentrated LP-5 protein was tested for its effect on the viability and cytotoxicity of various cancerous and non-cancerous cell lines using a cell counting kit-8 (CCK-8) assay. The LP-5 protein had the lowest IC50 value against the liver cancer line HepG2, at 58.15 μg/ml. Dose- and time-dependent morphological changes, such as cell shrinkage, blebbing formation, and cell fragmentation, were observed in treated cells. Apoptosis markers were evaluated using qPCR, flow cytometry, and western blot, and it was found that LP-5 protein increased the expression of Bax, Caspass-3, -8, -9, Cytochrome-C, and PARP, while decreasing the expression of Bcl2. Cell cycle arrest was also analyzed through qPCR, flow cytometry. Western blot results showed upregulated p21 and p27while CDK2, CDK4, CDK6, Cycline D1, and Cycline E1 were downregulated. These results suggest that LP-5 protein has potential as an anticancer agent against liver cancer cells.
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