Improvement of productivity of active horseradish peroxidase in Escherichia coli by coexpression of Dsb proteins
Improvement of productivity of active horseradish peroxidase in Escherichia coli by coexpression of Dsb proteins
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DOI：
10.1263/jbb.90.600
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发表时间：
2000-12-01
影响因子：
2.8
通讯作者：
Fukuda, H
中科院分区：
文献类型：
--
作者：
Kondo, A;Kohda, J;Fukuda, H
两类折叠辅助蛋白（分子伴侣蛋白和折叠酶）的共表达有望提高可溶性和活性重组蛋白的产量。本研究选择具有4个二硫键的辣根过氧化物酶（HRP）作为模型酶，在大肠杆菌中过表达。研究了一系列折叠辅助蛋白（DnaK、DnaJ、GrpE、GroEL/ES、触发因子（TF）、DsbA、DsbB、DsbC、DsbD和硫氧还蛋白（Trx））的共表达对大肠杆菌中活性HRP产量的影响。在37℃条件下，用1 μ m异丙基- β - d -硫代半乳糖苷（IPTG）诱导产生活性HRP，而用1 μ m IPTG诱导产生的活性HRP几乎可以忽略不计。共表达DsbA-DsbB （DsbAB）或DsbC-DsbD （DsbCD）可显著增加活性HRP的产生，而分子伴侣的共表达并不能提高活性HRP的产生。在HRP单表达系统中，1 mM IPTG诱导大肠杆菌细胞的生长明显受到抑制。相比之下，当HRP与DsbCD共表达时，没有观察到大肠杆菌的生长抑制。因此，Dsb蛋白的共表达促进了细胞生长和HRP的产量。
Coexpression of two classes of folding accessory proteins, molecular chaperones and foldases, can be expected to improve the productivity of soluble and active recombinant proteins. In this study, horseradish peroxidase (HRP), which has four disulfide bonds, was selected as a model enzyme and overexpressed in Escherichia coli. The effects of coexpression of a series of folding accessory proteins (DnaK, DnaJ, GrpE, GroEL/ES, trigger factor (TF), DsbA, DsbB, DsbC, DsbD, and thioredoxin (Trx)) on the productivity of active HRP in E, coli were examined. Active HRP was produced by very mild induction with 1 muM isopropyl-beta -D-thiogalactopyranoside (IPTG) at 37 degreesC, whereas the amount of active HRP produced by the induction with 1 mM IPTG was negligibly small. Active HRP production was increased significantly by coexpression of DsbA-DsbB (DsbAB) or DsbC-DsbD (DsbCD), while coexpression of molecular chaperones did not improve active HRP production. The growth of E. coli cells was inhibited significantly by the induction with 1 mM IPTG in a HRP single expression system. In contrast, when HRP was coexpressed with DsbCD, the growth inhibition of E. coli was not observed. Therefore, coexpression of Dsb proteins improves both the cell growth and the productivity of HRP.