Efficient production of extracellular proteins with Escherichia coli by means of optimized coexpression of bacteriocin release proteins.
Efficient production of extracellular proteins with Escherichia coli by means of optimized coexpression of bacteriocin release proteins.
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DOI：
10.1016/j.jbiotec.2009.11.019
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发表时间：
2010-02
影响因子：
4.1
通讯作者：
B. Sommer;K. Friehs;E. Flaschel
中科院分区：
文献类型：
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作者：
B. Sommer;K. Friehs;E. Flaschel
为了方便大肠杆菌产生的重组蛋白的可及性，可以通过细菌素释放蛋白（BRP）的共表达来实现细胞外表达。对不同类型的BRPs进行测试，以优化蛋白质分泌到培养基中。其中包括已被充分研究的Colicin E1和Cloacin DF13细菌素及其变体的BRPs。BRP的表达受到阿拉伯糖诱导的pbad启动子的严格控制，该启动子可以大范围调节表达强度。利用适当的阿拉伯糖浓度，确定了一个浓度范围，使模型蛋白碱性磷酸酶和β-内酰胺酶有效分泌，90-95%的蛋白释放到培养基中。对BRP表达和蛋白分泌的动力学研究显示，诱导后5-10min内细胞外蛋白浓度迅速升高。或者，在培养过程中微调BRP的表达，通过建立适当的诱导策略，蛋白质的产生和分泌可以解耦合，在达到最大生物量浓度后的3h内，高达90%的碱性磷酸酶被释放到培养基中。微调和生长解耦合的BRP表达均可解释细胞外碱性磷酸酶浓度约为500mg - 1的培养液和每克细胞干质量50mg酶的选择性。
Aiming to facilitate the accessibility of recombinant proteins produced with Escherichia coli, extracellular expression may be achieved by means of bacteriocin release protein (BRP) coexpression. Different types of BRPs were tested in order to optimize protein secretion into the culture medium. Those included the well-studied BRPs of the Colicin E1 and Cloacin DF13 bacteriocins and variants thereof. BRP expression was stringently controlled by means of the arabinose inducible PBADpromoter, which accounts for a broad-range adjustment of expression strength. Using appropriate arabinose concentrations, a concentration range was determined, that allowed efficient secretion of the model proteins alkaline phosphatase and β-lactamase, with 90–95% of the proteins released into the culture medium. Kinetic investigations into BRP expression and protein secretion revealed a rapid increase of extracellular protein concentration within 5–10min past induction. Alternatively to fine-tuned BRP expression during cultivation, protein production and secretion could be decoupled by establishment of appropriate induction strategies and up to 90% of alkaline phosphatase was released into the culture medium within 3h after reaching maximum biomasss concentrations. Both, fine-tuned and growth decoupled BRP expression accounted for extracellular alkaline phosphatase concentrations of roughly 500mgl−1of culture broth and selectivities of 50mg of this enzyme per gram of cell dry mass, respectively.