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Int. J. Mol. Sci. 2012, 13(5), 6246-6265; doi:10.3390/ijms13056246
Article

A Novel Apoptosis Correlated Molecule: Expression and Characterization of Protein Latcripin-1 from Lentinula edodes C91–3

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Received: 13 March 2012 / Revised: 9 May 2012 / Accepted: 11 May 2012 / Published: 21 May 2012
(This article belongs to the collection Programmed Cell Death and Apoptosis)
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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 Lentinula edodes; apoptosis; protein Latcripin-1; Pichia pastoris; expression
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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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.

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