Expression and in vitro anticancer activity of Lp16-PSP, a member of the YjgF/YER057c/UK114 protein family from the mush
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ORIGINAL PAPER
Expression and in vitro anticancer activity of Lp16‑PSP, a member of the YjgF/YER057c/UK114 protein family from the mushroom Lentinula edodes C91‑3 Thomson Patrick Joseph1,2 · Qianqian Zhao3 · Warren Chanda1 · Sadia Kanwal4 · Yukun Fang1 · MinTao Zhong1 · Min Huang1 Received: 21 March 2020 / Revised: 2 October 2020 / Accepted: 19 October 2020 © Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract Latcripin-16 (Lp16-PSP) is a gene that was extracted as a result of de novo characterization of the Lentinula edodes strain C91-3 transcriptome. The aim of the present study was to clone, express, and investigate the selective in vitro anticancer potential of Lp16-PSP in human cell lines. Lp16-PSP was analyzed using bioinformatics tools, cloned in a prokaryotic expression vector pET32a (+) and transformed into E. coli Rosetta gami. It was expressed and solubilized under optimized conditions. The differential scanning fluorometry (DSF)-guided refolding method was used with modifications to identify the proper refolding conditions for the Lp16-PSP protein. To determine the selective anticancer potential of Lp16-PSP, a panel of human cancerous and non-cancerous cell lines was used. Lp16-PSP protein was identified as endoribonuclease L-PSP protein and a member of the highly conserved YjgF/YER057c/UK114 protein superfamily. Lp16-PSP was expressed under optimized conditions (37 °C for 4 h following induction with 0.5 mM isopropyl β-d-1-thiogalactopyranoside). Solubilization was achieved with mild solubilization buffer containing 2 M urea using the freeze–thaw method. The DSF guided refolding method identified the proper refolding conditions (50 mM Tris–HCl, 100 mM NaCl, 1 mM EDTA, 400 mM Arginine, 0.2 mM GSH and 2 mM GSSG; pH 8.0) for Lp16-PSP, with a melting transition of ~ 58 °C. A final yield of ~ 16 mg of purified Lp16-PSP from 1 L of culture was obtained following dialysis and concentration by PEG 20,000. A Cell Counting Kit-8 assay revealed the selective cytotoxic effect of Lp16-PSP. The HL-60 cell line was demonstrated to be most sensitive to Lp16-PSP, with an I C50 value of 74.4 ± 1.07 µg/ml. The results of the present study suggest that Lp16-PSP may serve as a potential anticancer agent; however, further investigation is required to characterize this anticancer effect and to elucidate the molecular mechanism underlying the action of Lp16-PSP. Keywords Lentinula edodes C91-3 · Latcripin-16 · Endoribonuclease · HL-60 cell line Communicated by Erko Stackebrandt.
Introduction
* Min Huang [email protected]
Cancer is a global health problem with high morbidity and mortality worldwide. In the United States alone, ~ 1,806,590 new cases and ~ 606,520 cancer-associated mortalities were expected in 2020 (Siegel et al. 2020). Chemotherapy has been extensively used to treat various cancers; however, chemotherapeutic agents often cause severe side effects and cancers may become chemoresistant (Luqmani 2005). Therefore, the development of novel agents with no or minimal side effects is impo
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