Evaluation of pre-induction temperature, cell growth at induction and IPTG concentration on the expression of a leptospiral protein in E. coli using shaking flasks and microbioreactor.
Evaluation of pre-induction temperature, cell growth at induction and IPTG concentration on the expression of a leptospiral protein in E. coli using shaking flasks and microbioreactor.
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DOI：
10.1186/1756-0500-7-671
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发表时间：
2014-09-25
影响因子：
1.8
通讯作者：
Medeiros MA
中科院分区：
文献类型：
--
作者：
Larentis AL;Nicolau JF;Esteves Gdos S;Vareschini DT;de Almeida FV;dos Reis MG;Galler R;Medeiros MA
钩端螺旋体病是一种人畜共患病，在建成区日益流行，特别是在那些居住在卫生基础设施较差或根本不存在的不稳定住房中的社区。钩端螺旋体病可以致命，因为它的症状很容易与其他疾病的症状混淆。因此，需要快速诊断，以便有效治疗。使用钩端螺旋体免疫球蛋白样 (Lig) 蛋白诊断钩端螺旋体病的测试目前正在 Fiocruz 进行最终验证。在这项工作中，评估了 LigB (131-645aa) 在大肠杆菌 BL21 (DE3)Star™/pAE 中的表达过程。在两种不同预诱导温度（28℃和37℃）下的实验没有发现显着差异。然后，将菌株在 37°C 下培养直至添加 IPTG，然后在 28°C 下进行诱导，从而缩短了总处理时间。在此条件下，使用中心复合设计评估两个变量的表达：诱导 LigB (131-645aa) 的细胞生长（600 nm 处的吸光度在 0.75 和 2.0 之间）和诱导剂浓度（0.1 mM 至 1 mM IPTG）。这两个变量都会影响细胞生长和蛋白质表达。在 Absind = 2.0 的摇瓶中，在最终指数生长期进行诱导产生了更高的细胞浓度和 LigB (131-645aa) 生产力。 IPTG 浓度具有负面影响，可能比分子生物学常用浓度 (1 mM) 低十倍，同时保持相似水平的表达并减少对细胞生长的损害。 LigB (131-645aa) 的表达与细胞生长相关。还在微生物反应器中使用 0.1 mM IPTG 在 28°C 下进行指数期结束时的诱导 4 小时，达到更高的细胞密度和 970 mg/L 蛋白质。 LigB (131-645aa) 通过镍亲和层析纯化，同质性为 91%。通过实验设计，可以评估诱导变量对可溶性 LigB (131-645aa) 表达的影响和相互作用，以提高钩端螺旋体病诊断快速检测的生产效率并降低生产成本。
Leptospirosis is a zoonose that is increasingly endemic in built-up areas, especially where there are communities living in precarious housing with poor or non-existent sanitation infrastructure. Leptospirosis can kill, for its symptoms are easily confused with those of other diseases. As such, a rapid diagnosis is required so it can be treated effectively. A test for leptospirosis diagnosis using Leptospira Immunoglobulin-like (Lig) proteins is currently at final validation at Fiocruz. In this work, the process for expression of LigB (131-645aa) in E. coli BL21 (DE3)Star™/pAE was evaluated. No significant difference was found for the experiments at two different pre-induction temperatures (28°C and 37°C). Then, the strain was cultivated at 37°C until IPTG addition, followed by induction at 28°C, thereby reducing the overall process time. Under this condition, expression was assessed using central composite design for two variables: cell growth at which LigB (131-645aa) was induced (absorbance at 600 nm between 0.75 and 2.0) and inducer concentration (0.1 mM to 1 mM IPTG). Both variables influenced cell growth and protein expression. Induction at the final exponential growth phase in shaking flasks with Absind = 2.0 yielded higher cell concentrations and LigB (131-645aa) productivities. IPTG concentration had a negative effect and could be ten-fold lower than the concentration commonly used in molecular biology (1 mM), while keeping expression at similar levels and inducing less damage to cell growth. The expression of LigB (131-645aa) was associated with cell growth. The induction at the end of the exponential phase using 0.1 mM IPTG at 28°C for 4 h was also performed in microbioreactors, reaching higher cell densities and 970 mg/L protein. LigB (131-645aa) was purified by nickel affinity chromatography with 91% homogeneity. It was possible to assess the effects and interactions of the induction variables on the expression of soluble LigB (131-645aa) using experimental design, with a view to improving process productivity and reducing the production costs of a rapid test for leptospirosis diagnosis.