International Journal of Molecular Sciences (May 2012)
A Novel Apoptosis Correlated Molecule: Expression and Characterization of Protein Latcripin-1 from <em>Lentinula </em><em>e</em><em>dodes</em> C<sub>91–3</sub>
Abstract
An apoptosis correlated molecule—protein Latcripin-1 of <em>Lentinula</em><em> edodes</em> C<sub>91</sub><sub>-</sub><sub>3</sub>—was expressed and characterized in <em>Pichia pastoris</em> GS115. The total RNA was obtained from <em>Lentinula edodes</em> C<sub>91–3</sub>. 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 <em>Pichia pastoris</em> 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 (<em>p </em>< 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 (<em>p </em>< 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.
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