Chaperone Coexpression Plasmids: Differential and Synergistic Roles of DnaK-DnaJ-GrpE and GroEL-GroES in Assisting Folding of an Allergen of Japanese Cedar Pollen, Cryj2, inEscherichia coli
Chaperone Coexpression Plasmids: Differential and Synergistic Roles of DnaK-DnaJ-GrpE and GroEL-GroES in Assisting Folding of an Allergen of Japanese Cedar Pollen, Cryj2, inEscherichia coli
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DOI：
10.1128/aem.64.5.1694-1699.1998
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发表时间：
1998-05
影响因子：
4.4
通讯作者：
Kazuyo Nishihara;M. Kanemori;M. Kitagawa;H. Yanagi;T. Yura
中科院分区：
文献类型：
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作者：
Kazuyo Nishihara;M. Kanemori;M. Kitagawa;H. Yanagi;T. Yura
摘要构建了可用于控制表达DNAK-DNAJ-GRPE和/或GroEL-groes伴侣蛋白团队的载体，以便于评估这些伴侣蛋白团队对重组蛋白在大肠杆菌中折叠或组装的影响。当L-阿拉伯糖和四环素以剂量依赖的方式分别加入启动子时，获得的典型的pACYC184表达载体能够表达来自不同启动子的主要的DNAK-DNAJ-GRPE和GroEL-Groes伴侣。用来确定这个系统是否有用的模型蛋白质是日本雪松花粉的过敏原Cryj2，当它在E.coliK-12中产生时，它是不稳定的。在野生型和几个突变细菌中检测了伴侣蛋白共表达对Cryj2折叠、聚集和稳定性的影响。适当水平的DNAK-DNAJ-GRPE和/或GroEL-Groes伴侣蛋白的共表达可显著稳定和积累Cryj2，而不会引起广泛的聚集。用缺乏每一种伴侣蛋白(DNAK、DNAJ、GRPE、GroEL和GroES)或热休克转录因子ς32的突变体进行的实验表明，两个伴侣团队都关键地参与了Cryj2折叠，但它们以不同的方式参与。此外，还观察到在特定温度下，在没有ς32的情况下，两个伴侣团队在防止Cryj2聚集方面具有协同作用。
ABSTRACT Plasmids that can be used for controlled expression of the DnaK-DnaJ-GrpE and/or GroEL-GroES chaperone team were constructed in order to facilitate assessment of the effects of these chaperone teams on folding or assembly of recombinant proteins in Escherichia coli. A typical pACYC184-based plasmid which was obtained could express the major DnaK-DnaJ-GrpE and GroEL-GroES chaperone teams from separate promoters when l-arabinose and tetracycline, respectively, were added in a dose-dependent fashion. The model protein used to determine whether this system was useful was an allergen of Japanese cedar pollen, Cryj2, which was unstable when it was produced in E. coli K-12. The effects of chaperone coexpression on the folding, aggregation, and stability of Cryj2 were examined in the wild type and in several mutant bacteria. Coexpression of the DnaK-DnaJ-GrpE and/or GroEL-GroES chaperone team at appropriate levels resulted in marked stabilization and accumulation of Cryj2 without extensive aggregation. Experiments performed with mutants that lack each of the chaperone proteins (DnaK, DnaJ, GrpE, GroEL, and GroES) or heat shock transcription factor ς32 revealed that both chaperone teams are critically involved in Cryj2 folding but that they are involved in distinct ways. In addition, it was observed that the two chaperone teams have synergistic roles in preventing aggregation of Cryj2 in the absence of ς32 at certain temperatures.