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A Novel Apoptosis Correlated Molecule: Expression and Characterization of Protein Latcripin-1 from Lentinula edodes C91–3
Department of Microbiology, Dalian Medical University, No. 9 West Section, Lvshun South Road, Lvshunkou District, Dalian 116044, China
* Author to whom correspondence should be addressed.
Received: 13 March 2012; in revised form: 9 May 2012 / Accepted: 11 May 2012 / Published: 21 May 2012
Abstract: An apoptosis correlated molecule—protein Latcripin-1 of Lentinula edodes C91-3—was expressed and characterized in Pichia pastoris GS115. The total RNA was obtained from Lentinula edodes C91–3. According to the transcriptome, the full-length gene of Latcripin-1 was isolated with 3'-Full Rapid Amplification of cDNA Ends (RACE) and 5'-Full RACE methods. The full-length gene was inserted into the secretory expression vector pPIC9K. The protein Latcripin-1 was expressed in Pichia pastoris GS115 and analyzed by Sodium Dodecylsulfonate Polyacrylate Gel Electrophoresis (SDS-PAGE) and Western blot. The Western blot showed that the protein was expressed successfully. The biological function of protein Latcripin-1 on A549 cells was studied with flow cytometry and the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl-tetrazolium Bromide (MTT) method. The toxic effect of protein Latcripin-1 was detected with the MTT method by co-culturing the characterized protein with chick embryo fibroblasts. The MTT assay results showed that there was a great difference between protein Latcripin-1 groups and the control group (p < 0.05). There was no toxic effect of the characterized protein on chick embryo fibroblasts. The flow cytometry showed that there was a significant difference between the protein groups of interest and the control group according to apoptosis function (p < 0.05). At the same time, cell ultrastructure observed by transmission electron microscopy supported the results of flow cytometry. The work demonstrates that protein Latcripin-1 can induce apoptosis of human lung cancer cells A549 and brings new insights into and advantages to finding anti-tumor proteins.
Keywords: Lentinula edodes; apoptosis; protein Latcripin-1; Pichia pastoris; expression
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Liu, B.; Zhong, M.; Lun, Y.; Wang, X.; Sun, W.; Li, X.; Ning, A.; Cao, J.; Zhang, W.; Liu, L.; Huang, M. A Novel Apoptosis Correlated Molecule: Expression and Characterization of Protein Latcripin-1 from Lentinula edodes C91–3. Int. J. Mol. Sci. 2012, 13, 6246-6265.
Liu B, Zhong M, Lun Y, Wang X, Sun W, Li X, Ning A, Cao J, Zhang W, Liu L, Huang M. A Novel Apoptosis Correlated Molecule: Expression and Characterization of Protein Latcripin-1 from Lentinula edodes C91–3. International Journal of Molecular Sciences. 2012; 13(5):6246-6265.
Liu, Ben; Zhong, Mintao; Lun, Yongzhi; Wang, Xiaoli; Sun, Wenchang; Li, Xingyun; Ning, Anhong; Cao, Jing; Zhang, Wei; Liu, Lei; Huang, Min. 2012. "A Novel Apoptosis Correlated Molecule: Expression and Characterization of Protein Latcripin-1 from Lentinula edodes C91–3." Int. J. Mol. Sci. 13, no. 5: 6246-6265.