Optimizing HIV-1 protease production in Escherichia coli as fusion protein.
Optimizing HIV-1 protease production in Escherichia coli as fusion protein.
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DOI：
10.1186/1475-2859-10-53
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发表时间：
2011-06-30
影响因子：
6.4
通讯作者：
Pollegioni L
中科院分区：
文献类型：
--
作者：
Volontè F;Piubelli L;Pollegioni L
人类免疫缺陷病毒（HIV）是艾滋病及相关疾病的病原。由pol基因5'部分编码的天冬氨酸蛋白酶负责gag-pol多蛋白前体的蛋白水解加工，以产生成熟的衣壳蛋白以及逆转录酶和整合酶。HIV蛋白酶（HIV- 1pr）被认为是设计可用于治疗艾滋病的抑制剂的一个有吸引力的目标，因此它仍然是许多研究的对象。重组人免疫缺陷病毒1型蛋白酶（HIV-1Pr）在大肠杆菌细胞中作为细菌周质蛋白二硫醇氧化酶（DsbA）或谷胱甘肽s转移酶（GST）的融合蛋白过表达，也含有6组氨酸标签序列。通过设计合适的HIV-1Pr cDNA（用于大肠杆菌表达和避免自身蛋白水解），筛选6种不同的大肠杆菌菌株和5种生长培养基，优化蛋白表达。在大肠杆菌BL21-Codon Plus(DE3)-RIL宿主和添加1% （w/v）葡萄糖以最小化基础表达的TB或M9培养基中获得了最佳表达量。在测试的不同参数中，缓冲系统（基于磷酸盐）的存在和添加IPTG后37°C的生长温度对蛋白酶表达的增强起主要作用（高达10 mg嵌合DsbA:HIV-1Pr/L发酵液）。GST:HIVPr部分（50%）作为可溶性蛋白产生，而过表达的DsbA:HIV-1Pr嵌合蛋白大部分作为未加工的融合蛋白积累在包涵体中。在HiTrap螯合柱上开发了一种简单的再折叠程序，获得了可再折叠的DsbA:HIV-1Pr，回收率为80%。最后，溶出的DsbA:HIV-1Pr经肠激酶酶切后，每升发酵液中HIV-1Pr含量大于2 mg，纯度≤80%，而溶出的GST:HIVPr经肠激酶酶切后，每升发酵液中纯HIV-1Pr含量为~ 0.15 mg。通过使用这种优化的表达和纯化程序，可以在实验室规模上生产大量高质量的HIV-1Pr重组酶，从而用于进一步的生化研究。
Human immunodeficiency virus (HIV) is the etiological agent in AIDS and related diseases. The aspartyl protease encoded by the 5' portion of the pol gene is responsible for proteolytic processing of the gag-pol polyprotein precursor to yield the mature capsid protein and the reverse transcriptase and integrase enzymes. The HIV protease (HIV-1Pr) is considered an attractive target for designing inhibitors which could be used to tackle AIDS and therefore it is still the object of a number of investigations. A recombinant human immunodeficiency virus type 1 protease (HIV-1Pr) was overexpressed in Escherichia coli cells as a fusion protein with bacterial periplasmic protein dithiol oxidase (DsbA) or glutathione S-transferase (GST), also containing a six-histidine tag sequence. Protein expression was optimized by designing a suitable HIV-1Pr cDNA (for E. coli expression and to avoid autoproteolysis) and by screening six different E. coli strains and five growth media. The best expression yields were achieved in E. coli BL21-Codon Plus(DE3)-RIL host and in TB or M9 medium to which 1% (w/v) glucose was added to minimize basal expression. Among the different parameters assayed, the presence of a buffer system (based on phosphate salts) and a growth temperature of 37°C after adding IPTG played the main role in enhancing protease expression (up to 10 mg of chimeric DsbA:HIV-1Pr/L fermentation broth). GST:HIVPr was in part (50%) produced as soluble protein while the overexpressed DsbA:HIV-1Pr chimeric protein largely accumulated in inclusion bodies as unprocessed fusion protein. A simple refolding procedure was developed on HiTrap Chelating column that yielded a refolded DsbA:HIV-1Pr with a > 80% recovery. Finally, enterokinase digestion of resolubilized DsbA:HIV-1Pr gave more than 2 mg of HIV-1Pr per liter of fermentation broth with a purity ≤ 80%, while PreScission protease cleavage of soluble GST:HIVPr yielded ~ 0.15 mg of pure HIV-1Pr per liter. By using this optimized expression and purification procedure fairly large amounts of good-quality HIV-1Pr recombinant enzyme can be produced at the lab-scale and thus used for further biochemical studies.
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影响因子：
5.6
作者：
EGAN, SM;SCHLEIF, RF
通讯作者：
SCHLEIF, RF
影响因子：
3.2
作者：
Haldimann, A;Daniels, LL;Wanner, BL
通讯作者：
Wanner, BL
DOI：
10.1007/bf01028194
发表时间：
1993-06-01
期刊：
JOURNAL OF PROTEIN CHEMISTRY
影响因子：
--
作者：
HUI, JO;TOMASSELLI, AG;HEINRIKSON, RL
通讯作者：
HEINRIKSON, RL
影响因子：
5
作者：
Komai, T;Ishikawa, Y;Handa, H
通讯作者：
Handa, H
影响因子：
3.5
作者：
Volonte, Federica;Pollegioni, Loredano;Piubelli, Luciano
通讯作者：
Piubelli, Luciano