Ameer Khusro, Chirom Aarti, Paul Agastian
Research Department of Plant Biology and Biotechnology, Loyola College (Affiliated to University of Madras), Nungambakkam, Chennai, 600034, Tamil Nadu, India.
Research Department of Plant Biology and Biotechnology, Loyola College (Affiliated to University of Madras), Nungambakkam, Chennai, 600034, Tamil Nadu, India.
J Pharm Biomed Anal. 2020 Apr 15;182:113136. doi: 10.1016/j.jpba.2020.113136. Epub 2020 Jan 31.
The present study was investigated to purify and characterize anti-tubercular and anticancer protein from Staphylococcus hominis strain MANF2 under mild stress condition of Mentha piperita L. Initially, the in vitro anti-tubercular activity of strain MANF2 was determined against Mycobacterium tuberculosis H37Rv using luciferase reporter phage (LRP) assay which showed relative light unit reduction (RLU) of >90 %. Further, MTT test revealed promising in vitro anticancer trait of strain MANF2 against lung (A549) and colon (HT-29) cancer cell lines. Mild stress of M. piperita L. was provided to strain MANF2 at lag and log phase of its growth and the protein production was optimized statistically using central composite design of response surface methodology. Results showed enhanced protein production in the medium containing yeast extract (0.5 % w/v) and glycerol (1.5 % v/v), being supplemented with M. piperita L. (1.5 % v/v at log phase of strain MANF2. Protein was purified using standard purification techniques and showed single homogeneous band on SDS-PAGE with nominal molecular mass of 51293 Da, as confirmed by MALDI-TOF MS/MS. The N- amino acid sequencing showed homology with proline dehydrogenase (ProDH), thus, the protein was proposed to be new ProDH-like protein in S. hominis. Further, LRP test revealed concentration dependent (10-50 μg/mL) in vitro anti-tubercular properties of purified protein with significant RLU reductions of 36.8 ± 0.3-78.5 ± 0.4 %. The IC values of purified protein against A549 and HT-29 cancer cells were calculated as 42.2 and 48.4 μg/mL, respectively. In conclusion, protein purified from strain MANF2 under mild stress of M. piperita L can certainly be implied as efficacious anti-tubercular and anticancer agents in future.
本研究旨在从人葡萄球菌菌株MANF2中，在薄荷（Mentha piperita L.）轻度胁迫条件下纯化并鉴定抗结核和抗癌蛋白。最初，使用荧光素酶报告噬菌体（LRP）测定法测定了菌株MANF2对结核分枝杆菌H37Rv的体外抗结核活性，结果显示相对光单位减少（RLU）>90%。此外，MTT试验显示菌株MANF2对肺癌（A549）和结肠癌细胞系（HT-29）具有良好的体外抗癌特性。在菌株MANF2生长的延迟期和对数期对其施加薄荷的轻度胁迫，并使用响应面方法的中心复合设计对蛋白质产量进行统计学优化。结果表明，在含有酵母提取物（0.5% w/v）和甘油（1.5% v/v）的培养基中，添加薄荷（在菌株MANF2对数期为1.5% v/v）时蛋白质产量增加。使用标准纯化技术对蛋白质进行纯化，SDS-PAGE显示为单一均匀条带，经MALDI-TOF MS/MS确认，标称分子量为51293 Da。N-氨基酸测序显示与脯氨酸脱氢酶（ProDH）具有同源性，因此，该蛋白质被认为是人葡萄球菌中一种新的ProDH样蛋白。此外，LRP试验显示纯化蛋白具有浓度依赖性（10-50μg/mL）的体外抗结核特性，RLU显著降低36.8±0.3-78.5±0.4%。纯化蛋白对A549和HT-29癌细胞的IC值分别计算为42.2和48.4μg/mL。总之，在薄荷轻度胁迫下从菌株MANF2中纯化的蛋白质未来肯定可作为有效的抗结核和抗癌药物。
