Satheeshkumar Padikara K, Anu Prasannan V, Junaida Mohmed I, Madanan Madathiparambil G, Jebasingh Tennison, Nair Ananthakrishnan J, Nair Gangaprasad A, Nair Govinda Pillai M, Sudhakaran Perumana R
Interuniversity Centre for Genomics and Gene Technology, Department of Biotechnology, University of Kerala, Trivandrum, Kerala, India.
Regional Medical Research Centre (ICMR), Port Blair, Andaman and Nicobar Islands, India.
J Genet Eng Biotechnol. 2018 Dec;16(2):393-398. doi: 10.1016/j.jgeb.2018.01.004. Epub 2018 Feb 2.
Heterologous expression of Integral Membrane Proteins (IMPs) is reported to be toxic to the host system in many studies. Even though there are reports on various concerns like transformation efficiency, growth properties, protein toxicity, inefficient expression and protein degradation in IMP overexpression, no studies so far addressed these issues in a comprehensive way. In the present study, two transmembrane proteins of the pathogen , namely Signal peptidase (SP), and Leptospira Endostatin like A (Len-A) were taken along with a cytosolic protein Hydrolase (HYD) to assess the differences in transformation efficiency, protein toxicity, and protein stability when over expressed in (). Bioinformatics analysis to predict the transmembrane localization indicated that both SP and Len are targeted to the membrane. The three proteins were expressed in full length in the expression strain, BL 21 (DE3). Significant changes were observed for the strains transformed with IMP genes under the parameters analysed such as, the transformation efficiency, survival of colonies on IPTG-plate, culture growth kinetics and protein expression compared to the strain harbouring the cytosolic protein gene.
在许多研究中，据报道整合膜蛋白（IMPs）的异源表达对宿主系统有毒性。尽管有关于各种问题的报道，如转化效率、生长特性、蛋白质毒性、IMP过表达时表达效率低下和蛋白质降解等，但迄今为止尚无研究全面解决这些问题。在本研究中，选取了病原体的两种跨膜蛋白，即信号肽酶（SP）和类钩端螺旋体内皮抑素A（Len-A），以及一种胞质蛋白水解酶（HYD），以评估它们在（）中过表达时在转化效率、蛋白质毒性和蛋白质稳定性方面的差异。预测跨膜定位的生物信息学分析表明，SP和Len均靶向膜。这三种蛋白质在表达菌株BL 21（DE3）中全长表达。与携带胞质蛋白基因的菌株相比，在所分析的参数下，如转化效率、IPTG平板上菌落的存活率、培养生长动力学和蛋白质表达等方面，用IMP基因转化的菌株观察到了显著变化。
