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首页> 外文期刊>Iranian Journal of Microbiology >Cloning, optimization of induction conditions and purification of Mycobacterium tuberculosis Rv1733c protein expressed in Escherichia coli
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Cloning, optimization of induction conditions and purification of Mycobacterium tuberculosis Rv1733c protein expressed in Escherichia coli

机译：大肠杆菌表达的结核分枝杆菌Rv1733c蛋白的克隆，优化诱导条件及纯化

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Background and Objectives: Rv1733c is a latency antigen from Mycobacterium tuberculosis, a probable integral-membrane protein with promiscuous T-cell and B-cell epitopes, making it a potential vaccine candidate against tuberculosis. This study aimed to clone and optimize the expression of recombinant Rv1733c in Escherichia coli for purification. Materials and Methods: Chemically synthesized rv1733c coding sequence was cloned in pET-23a(+) followed by transforming E. coli BL21 (DE3) cells. To evaluate the induction conditions for optimized expression, factorial design of experiments was employed using four different media as well as four levels of isopropyl-b-D-thiogalactopyranosid [IPTG] concentration and duration of induction. The recombinant protein was then purified using a His-tag purification kit and detected through western blotting.Results: Recombinant Rv1733c (> 24 kDa) was expressed and accumulated in the cytoplasm of the E. coli cells. Medium composition showed the most significant effect on the yield of the recombinant protein (P = 0.000). The highest yield of recombinant Rv1733c occurred in the presence of 0.4 mM of IPTG in Terrific Broth medium (containing 1.2% tryptone, 2.4% yeast extract, 72 mM K2HPO4, 17 mM KH2PO4 and 0.4% glycerol) after 10 h at 37°C. Under these conditions, the expression level was around 0.5 g/L of culture medium. Purified Rv1733c was detected by an anti-polyhistidine antibody and a tuberculosis patient’s serum. Systematic optimization of induction conditions gave us high yield of recombinant polyhistidine-tagged Rv1733c in E. coli which was successfuly purified.Conclusion: We believe that the purified Rv1733c recombinant protein from M. tuberculosis might be a good candidate for vaccine production against tuberculosis.
机译：背景与目的：Rv1733c是结核分枝杆菌的潜伏期抗原，它是一种可能的整合膜蛋白，具有混杂的T细胞和B细胞抗原决定簇，使其成为抗结核疫苗的潜在候选者。这项研究旨在克隆和优化重组Rv1733c在大肠杆菌中的表达以进行纯化。材料和方法：将化学合成的rv1733c编码序列克隆到pET-23a（+）中，然后转化大肠杆菌BL21（DE3）细胞。为了评估优化表达的诱导条件，采用了因子分解实验设计，使用了四种不同的培养基以及四种水平的异丙基-b-D-硫代吡喃半乳糖苷[IPTG]浓度和诱导时间。然后用His-tag纯化试剂盒纯化重组蛋白，并通过western blotting检测。结果：重组Rv1733c（> 24kDa）在大肠杆菌细胞质中表达并积累。培养基组成对重组蛋白的产量显示出最显着的影响（P = 0.000）。重组Rv1733c的最高产量出现在37°C下10小时后的0.4％IPTG在Terrific Broth培养基（含有1.2％胰蛋白,、 2.4％酵母提取物，72 mM K2HPO4、17 mM KH2PO4和0.4％甘油）的存在下。在这些条件下，表达水平约为0.5 g / L的培养基。纯化的Rv1733c由抗多组氨酸抗体和结核病患者的血清检测到。诱导条件的系统优化使我们在大肠杆菌中成功纯化了带有多聚组氨酸标签的Rv1733c重组质粒。

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