Department of Chemical Engineering, Systems Biology and Tissue Engineering Research Center, National Chung Cheng University, Chiayi 621, Taiwan.
Department of Chemical Engineering, Systems Biology and Tissue Engineering Research Center, National Chung Cheng University, Chiayi 621, Taiwan.
J Biosci Bioeng. 2014 Sep;118(3):242-8. doi: 10.1016/j.jbiosc.2014.02.012. Epub 2014 Mar 18.
A co-expression system was established in Escherichia coli for enhancing the cellular expression of heat shock transcription factor, sigma 32 (σ(32)). A Shine-Dalgarno sequence and the rpoH gene of E. coli, which encodes σ(32), were cloned into a bacterial plasmid containing a gene fusion encoding a doubly tagged N-acetyl-d-neuraminic acid aldolase (GST-Neu5Ac aldolase-5R). After the IPTG induction, a substantially higher level of sigma 32 was observed up to 3 h in the co-expression cells, but an enhancement in the solubility of target protein was manifest only in the first hour. Nevertheless, the co-expression of sigma 32 led to higher level of Neu5Ac aldolase enzymatic activity in both the soluble and insoluble (inclusion body) fractions. The Neu5Ac aldolase activity of the supernatant from the lysate of cells co-expressing GST-Neu5Ac aldolase-5R and recombinant σ(32) was 3.4-fold higher at 3 h postinduction than that in cells overexpressing GST-Neu5Ac aldolase-5R in the absence of recombinantly expressed σ(32). The results of acrylamide quenching indicated that the conformational quality of the fusion protein was improved by the co-expression of recombinant σ(32). Thus, the increased level of intracellular σ(32) might have created favorable conditions for the proper folding of recombinant proteins through the cooperative effects of chaperones/heat shock proteins expressed by the E. coli host, which resulted in smaller inclusion bodies, improved conformational quality and a higher specific activity of the overexpressed GST-Neu5Ac aldolase-5R protein.
建立了大肠杆菌中的共表达系统，以增强热休克转录因子σ32（σ（32））的细胞表达。将大肠杆菌的 Shine-Dalgarno 序列和 rpoH 基因（编码σ（32））克隆到含有编码双标记 N-乙酰-d-神经氨酸醛缩酶（GST-Neu5Ac 醛缩酶-5R）的基因融合的细菌质粒中。在 IPTG 诱导后，在共表达细胞中观察到高达 3 h 的σ32 水平显著提高，但仅在第 1 小时观察到靶蛋白的可溶性增强。然而，共表达σ32导致可溶性和不溶性（包涵体）部分中 Neu5Ac 醛缩酶酶活性水平更高。在共表达 GST-Neu5Ac 醛缩酶-5R 和重组σ（32）的细胞裂解物上清液中的 Neu5Ac 醛缩酶活性在诱导后 3 h 时比在不存在重组表达的σ（32）的情况下过表达 GST-Neu5Ac 醛缩酶-5R 的细胞高 3.4 倍。丙烯酰胺猝灭的结果表明，融合蛋白的构象质量通过共表达重组σ（32）得到改善。因此，细胞内σ（32）水平的增加可能通过大肠杆菌宿主表达的伴侣蛋白/热休克蛋白的协同作用为重组蛋白的正确折叠创造了有利条件，导致包涵体更小、构象质量更好和 GST-Neu5Ac 醛缩酶-5R 蛋白的比活性更高。
