The Protocatechuate 3,4-Dioxygenase Solubility (PCDS) Tag Enhances the Expression and Solubility of Heterogenous Proteins in Escherichia coli.
The Protocatechuate 3,4-Dioxygenase Solubility (PCDS) Tag Enhances the Expression and Solubility of Heterogenous Proteins in Escherichia coli.
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原儿茶酸 3,4-双加氧酶溶解度 (PCDS) 标签增强异源蛋白在大肠杆菌中的表达和溶解度
DOI：
10.3389/fmicb.2021.779541
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发表时间：
2021
影响因子：
5.2
通讯作者：
Chao HJ
中科院分区：
文献类型：
--
作者：
Zou L;Li S;Li N;Ruan SL;Chen J;Wu J;Yan D;Chao HJ
大肠杆菌已被开发为重组蛋白表达的最常见宿主。不幸的是，仍然有一些蛋白质对大肠杆菌中高水平的异源可溶性表达具有抵抗力。蛋白质和肽融合标签是增加靶蛋白表达的最重要方法之一，似乎也会影响表达效率和溶解度。在这项研究中，我们鉴定了一种短的 15 个残基增强溶解度肽，PCDS（原儿茶酸 3,4-双加氧酶溶解度）标签，它增强了大肠杆菌中异源蛋白的表达。该 PCDS 标签是一段 45 bp 长的序列，编码参与原儿茶酸 3,4-双加氧酶可溶性表达的肽标签，由恶臭假单胞菌 NCIMB 9866 的 pcaHG98 基因编码。该 45 bp 序列也有利于 pcaHG98 基因扩增。该标签被证明对于 PcaHG98 在大肠杆菌中异源可溶性表达是必需的。纯化的 His6-PcaHG98e04-PCDS 对原儿茶酸作为底物表现出 205.63±14.23U/mg 的活性，并且该活性不受 PCDS 标签的影响。该 PCDS 标签已与哺乳动物黄色荧光蛋白 (YFP) 融合，构建无终止密码子的 YFP-PCDS 和带有终止密码子的 YFPt-PCDS。与单独的YFP相比，YFP-PCDS和YFPt-PCDS的总蛋白表达分别显着扩增至1.6倍和2倍。因此，与相同条件下表达的His6-YFP相比，His6-YFP-PCDS和His6-YFPt-PCDS的可溶性蛋白产量分别高1.6倍和3倍。 His6-YFP、His6-YFP-PCDS 和 His6-YFPt-PCDS 也显示出一致的荧光发射光谱，在 400 至 700 nm 的扫描范围内峰值位于 530 nm。这些结果表明，使用PCDS标签是提高大肠杆菌中异源蛋白表达的有效方法。
Escherichia coli has been developed as the most common host for recombinant protein expression. Unfortunately, there are still some proteins that are resistant to high levels of heterologous soluble expression in E. coli. Protein and peptide fusion tags are one of the most important methods for increasing target protein expression and seem to influence the expression efficiency and solubility as well. In this study, we identify a short 15-residue enhancing solubility peptide, the PCDS (protocatechuate 3,4-dioxygenase solubility) tag, which enhances heterologous protein expression in E. coli. This PCDS tag is a 45-bp long sequence encoding a peptide tag involved in the soluble expression of protocatechuate 3,4-dioxygenase, encoded by the pcaHG98 genes of Pseudomonas putida NCIMB 9866. The 45-bp sequence was also beneficial for pcaHG98 gene amplification. This tag was shown to be necessary for the heterologous soluble expression of PcaHG98 in E. coli. Purified His6-PcaHG98e04-PCDS exhibited an activity of 205.63±14.23U/mg against protocatechuate as a substrate, and this activity was not affected by a PCDS tag. This PCDS tag has been fused to the mammalian yellow fluorescent protein (YFP) to construct YFP-PCDS without its termination codons and YFPt-PCDS with. The total protein expressions of YFP-PCDS and YFPt-PCDS were significantly amplified up to 1.6-fold and 2-fold, respectively, compared to YFP alone. Accordingly, His6-YFP-PCDS and His6-YFPt-PCDS had 1.6-fold and 3-fold higher soluble protein yields, respectively, than His6-YFP expressed under the same conditions. His6-YFP, His6-YFP-PCDS, and His6-YFPt-PCDS also showed consistent fluorescence emission spectra, with a peak at 530nm over a scanning range from 400 to 700nm. These results indicated that the use of the PCDS tag is an effective way to improve heterologous protein expression in E. coli.
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DOI：
10.1016/s0021-9673(00)93969-4
发表时间：
1987-12-18
期刊：
JOURNAL OF CHROMATOGRAPHY
影响因子：
--
作者：
HOCHULI, E;DOBELI, H;SCHACHER, A
通讯作者：
SCHACHER, A
DOI：
10.1038/nbt1088-1204
发表时间：
1988-10-01
期刊：
BIO-TECHNOLOGY
影响因子：
--
作者：
HOPP, TP;PRICKETT, KS;CONLON, PJ
通讯作者：
CONLON, PJ
DOI：
10.1385/1-59259-148-5:167
发表时间：
2001-01-01
期刊：
MENINGOCOCCAL VACCINES
影响因子：
--
作者：
Christodoulides, M;Jolley, KA;Heckels, JE
通讯作者：
Heckels, JE
影响因子：
14.9
作者：
Ferrando, A;Koncz-Kálmán, Z;Koncz, C
通讯作者：
Koncz, C
影响因子：
8
作者：
Kapust, RB;Waugh, DS
通讯作者：
Waugh, DS