A novel method for promoting heterologous protein expression in Escherichia coli by fusion with the HIV-1 TAT core domain
A novel method for promoting heterologous protein expression in Escherichia coli by fusion with the HIV-1 TAT core domain
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一种通过与 HIV-1 TAT 核心结构域融合促进大肠杆菌异源蛋白表达的新方法
DOI：
10.1007/s00726-010-0534-2
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发表时间：
2010-08-01
期刊：
影响因子：
3.5
通讯作者：
Zhang, Chenggang
中科院分区：
文献类型：
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作者：
Wu, Yonghong;Ren, Changhong;Zhang, Chenggang
人类免疫缺陷病毒1型（HIV-1）转录反式激活因子（达特）蛋白是蛋白转导结构域（PTD）超家族的成员，可以将异源蛋白递送到大多数生物膜上而不丧失生物活性。然而，关于包含序列“YGRKKRRQRRR”的达特核心结构域是否具有其他功能，目前还没有报道。由于达特核心结构域是最碱性的（pI = 12.8）并且具有生物膜穿越能力，我们推测它可能由于靶蛋白在细胞中的亚细胞再分布而影响蛋白表达水平。为了解决这一问题，我们构建了含核心区编码区的原核表达载体pET 28 b-TAT-EGFP（以pET 28 b-EGFP为对照），并转化E.以异丙基-β-d-硫代半乳糖苷（IPTG）为诱导剂，在大肠杆菌BL 21（DE 3）中表达增强型绿色荧光蛋白（EGFP）。用15% SDS-PAGE分离等量的总蛋白，用Western blot鉴定，用Southern blot检测质粒拷贝数。为了进一步研究异源蛋白在大肠杆菌中的亚细胞定位。coli细胞，用氯仿和渗透压休克技术提取细胞质和周质组分。有趣的是，我们的数据表明达特核心结构域不仅能够促进异源蛋白在大肠杆菌中的表达。大肠杆菌中，TAT核心结构域不仅提高了外源蛋白的产量，而且提高了外源蛋白的可溶性，而达特核心结构域对含TAT克隆和不含TAT克隆的质粒拷贝数没有影响。此外，TAT标记蛋白主要定位于细胞质中，并随着表达时间的延长，沿着在周质间隙中积累，而无TAT标记蛋白主要表达于周质中，细胞质中表达量较少。对表达蛋白在细胞质和周质中的分布的进一步研究表明，达特核心结构域可能最初促进蛋白在细胞质中的表达，然后以浓度依赖性的方式将它们部分地穿过细胞膜递送到周质空间。综上所述，我们的研究结果为利用达特核心结构域提高异源蛋白在原核细胞中的表达提供了一种新的方法，为蛋白质工程中生物活性蛋白的表达提供了新的途径。
The human immunodeficiency virus type 1 (HIV-1) transactivator of transcription (TAT) protein, a member of the protein transduction domain (PTD) superfamily, can deliver heterologous proteins across most biomembranes without losing bioactivity. However, there is no report on whether the TAT core domain containing the sequence ‘YGRKKRRQRRR’ has other functions. As the TAT core domain is most basic (pI = 12.8) and has biomembrane crossing ability, we hypothesized it might probably influence the protein expression level due to subcellular redistribution of target proteins in the cells. To address this issue, we constructed the prokaryotic expression vector pET28b-TAT-EGFP (using the vector pET28b-EGFP for control) containing the core domain coding region, and transformed the vector into E. coli BL21 (DE3) cells for expression of the enhanced green fluorescent protein (EGFP) with the inducer isopropyl-β-d-thiogalactopyranoside (IPTG). Equal amount of the total proteins were fractionated using 15% SDS-PAGE and identified by western blot, and the plasmid copy number was assayed by Southern blot. In order to further study the subcellular localization of heterologous proteins in E. coli cells, the cytoplasmic and periplasmic components were extracted by chloroform and osmotic shock techniques. Interestingly, our data showed that the TAT core domain was not only able to promote the heterologous protein expression in E. coli, but also improve the yields and the solubility of heterologous proteins, while the plasmid copy number of TAT-containing clones and TAT-free clones was not affected by the TAT core domain. In addition, the TAT-tagged protein was mainly localized in the cytoplasm and also accumulated in the periplasmic space along with the time for protein expression, while in contrast, the TAT-free protein was mainly expressed in the periplasm and only a few in cytoplasm. A further examination on the distribution of the expressed proteins in cytoplasm and periplasm suggested that the TAT core domain might promote protein expression in the cytoplasm initially and then partially deliver them across the cytomembrane to the periplasmic space in a concentration-dependent manner. Taken together, our current data have provided a novel method for improving heterologous protein expression in prokaryotic cells by fusion with the TAT core domain, which will promote expression efficiency of bioactive proteins for protein engineering.