title: Expression and characterization of protein Latcripin-3, an antioxidant and antitumor molecule from Lentinula edodes C91-3.
abstract: In this study, an anti-oxidant and anti-tumor protein Latcripin-3 of Lentinula edodes C91-3 was expressed in Escherichia coli. for the first time. According to the cDNA library, the full-length gene of Latcripin-3 was cloned by the methods of 3'-full rapid amplification of cDNA Ends (RACE) and 5'-full RACE. The structural domain gene of Latcripin-3 was inserted into the pET32 a(+). The functional protein of Latcripin-3 was expressed in Rosetta-gami (DE3) E. coli, evaluated by Western blotting and mass spectrometry. DPPH testing showed that the protein Latcripin-3 can scavenge free radicals remarkably well. The activity of functional protein Latcripin-3 on A549 cells was studied with flow cytometry and the MTT method. The MTT assay results showed that there was a decreases in cell viability in a dose-dependent and time-dependent manner in protein Latcripin-3 treated groups. Flow cytometry demonstrated that Latcripin-3 can induce apoptosis and block S phase dramatically in human A549 lung cancer cells as compared to the control group. At the same time, the cell ultrastructure observed by transmission electron microscopy supported the results of flow cytometry. This research offers new insights and advantages for identifying anti-oxidant and anti-tumor proteins.
authors.affiliation: Department of Microbiology, Dalian Medical University, Dalian, LiaoNing, China E-mail : huangminchao@163.com.
authors.forename: Xiao-Hua
authors.initials: XH
authors.lastname: Ann
authors.forename: Yong-Zhi
authors.initials: YZ
authors.lastname: Lun
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Ben
authors.initials: B
authors.lastname: Liu
authors.forename: Xing-Yun
authors.initials: XY
authors.lastname: Li
authors.forename: Min-Tao
authors.initials: MT
authors.lastname: Zhong
authors.forename: Xiao-Li
authors.initials: XL
authors.lastname: Wang
authors.forename: Jing
authors.initials: J
authors.lastname: Cao
authors.forename: An-Hong
authors.initials: AH
authors.lastname: Ning
authors.forename: Min
authors.initials: M
authors.lastname: Huang
mesh_terms: D000230:Adenocarcinoma / Q000378:metabolism / Q000473:pathology; D000970:Antineoplastic Agents / Q000378:metabolism*; D000975:Antioxidants / Q000378:metabolism*; D017209:Apoptosis; D015153:Blotting, Western; D002453:Cell Cycle; D049109:Cell Proliferation; D005434:Flow Cytometry; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism* / Q000473:pathology; D020942:Shiitake Mushrooms / Q000737:chemistry*; D014407:Tumor Cells, Cultured
chemical_list: D000970:Antineoplastic Agents; D000975:Antioxidants; D005656:Fungal Proteins; C000595217:Latcripin-13 protein, Lentinula edodes