Kinetics of heat-shock response and inclusion body formation during temperature-induced production of basic fibroblast growth factor in high-cell-density cultures of recombinant Escherichia coli
Kinetics of heat-shock response and inclusion body formation during temperature-induced production of basic fibroblast growth factor in high-cell-density cultures of recombinant Escherichia coli
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DOI：
10.1021/bp0000959
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发表时间：
2000-11-01
影响因子：
2.9
通讯作者：
Rinas, U
中科院分区：
文献类型：
--
作者：
Hoffmann, F;Rinas, U
在葡萄糖限制的高细胞密度培养物中研究了重组大肠杆菌TG 1中热休克反应和包涵体形成的动力学，以响应温度诱导的人碱性成纤维细胞生长因子（hFGF-2）的产生，该蛋白质部分聚集成包涵体。热休克蛋白的最大合成速率与使用携带无诱导结构基因的表达载体的菌株的对照培养中的那些相似。然而，最大的诱导许多热休克蛋白，包括DnaK，ClpB，和HtpG至少30分钟后达到合成的hFGF-2时，同时诱导的温度上移。在该第一生产阶段期间，hFGF-2仅沉积在不溶性细胞级分中。此后，也观察到可溶性hFGF-2的积累，表明重组蛋白需要热休克分子伴侣在升高的温度下正确折叠。即使在野生型dnaK（+）背景下，强重组蛋白的产生也延长了大多数热休克蛋白（包括GroELS、DnaK、ClpB和HtpG）的合成。与此相反，小的热休克蛋白IbpA和IbpB的合成率下降1小时内的preinduction值在控制和hFGF-2生产的文化。在生产培养中，当可溶性hFGF-2开始积累时，IbpA和IbpB的合成停止到不可检测的水平，而其他热休克蛋白的合成速率，包括属于DnaK和GroEL家族的那些，在整个生产阶段保持高水平。
The kinetics of the heat-shock response and the formation of inclusion bodies in recombinant Escherichia coli TG1 were studied in glucose-limited high-cell-density cultures in response to temperature-induced production of human basic fibroblast growth factor (hFGF-2), a protein which partially aggregates into inclusion bodies. The maximum synthesis rates of heat-shock proteins were similar to those in a control cultivation with a strain carrying an expression vector without inducible structural gene. However, the maximum of induction for many heat-shock proteins including DnaK, ClpB, and HtpG was reached at least 30 min later when synthesis of hFGF-2 was simultaneously induced by the temperature upshift. During this first production phase, hFGF-2 was exclusively deposited in the insoluble cell fraction. Thereafter, accumulation of soluble hFGF-2 was observed, too, indicating that the recombinant protein needs heat-shock chaperones for proper folding at elevated temperatures. Strong recombinant protein production prolonged the synthesis of the majority of heat-shock proteins (including GroELS, DnaK, ClpB, and HtpG) even in a wildtype dnaK(+) background. In contrast, the synthesis rates of the small heat-shock proteins IbpA and IbpB declined within 1 h to preinduction values in control and hFGF-2 producing cultures. In the producing cultivation, IbpA and IbpB synthesis ceased to an undetectable level when soluble hFGF-2 started to accumulate, whereas the synthesis rates of the other heat-shock proteins including those belonging to the DnaK and GroEL families remained high throughout the entire production phase.