In vivo polyester immobilized sortase for tagless protein purification.
In vivo polyester immobilized sortase for tagless protein purification.
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DOI：
10.1186/s12934-015-0385-3
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发表时间：
2015-11-25
影响因子：
6.4
通讯作者：
Rehm BH
中科院分区：
文献类型：
--
作者：
Hay ID;Du J;Reyes PR;Rehm BH
实验室规模的重组蛋白生产和纯化技术往往很复杂，涉及多个层析步骤和专门的设备和试剂。本研究证明，在大肠杆菌中，通过将聚羟基烷酸(PHA)与聚酯合酶基因(Phac)进行基因融合，可以将重组蛋白以共价方式固定在聚羟基烷酸(PHA)微珠的表面。在合成酶和目的蛋白之间插入一个自切割模块、一个来自金黄色葡萄球菌的修饰的排序酶A(SrtA)及其5个氨基酸识别序列，从而使蛋白质的生产和纯化变得简单。编码三方PHAC-SrtA-Target融合蛋白的杂合基因的产生，使PHA珠体表面感兴趣的蛋白能够在体内固定。经过简单的细胞裂解和PHA小球的分离，通过CaCl2和甘氨酸激活索酸酶，可以选择性和有效地将目的蛋白从小球中释放出来。未经优化，一步可分离出纯度约为98%的可溶性蛋白质，纯度可达6 mg/L。该方法被用来生产和分离三种蛋白质：绿色荧光蛋白、麦芽糖结合蛋白和结核分枝杆菌候选疫苗Rv1626。我们开发了一种易于生产和纯化重组蛋白的新技术。该技术能够在不到2天的时间内生产和纯化适合研究应用的高产率蛋白质。不需要昂贵的或专门的蛋白质层析设备、树脂、试剂或专业知识。本文的在线版本(doi：10.1186/s12934-0150385-3)包含补充材料，授权用户可以使用。
Laboratory scale recombinant protein production and purification techniques are often complicated, involving multiple chromatography steps and specialized equipment and reagents. Here it was demonstrated that recombinant proteins can be expressed as covalently immobilized to the surface of polyester (polyhydroxyalkanoate, PHA) beads in vivo in Escherichia coli by genetically fusing them to a polyester synthase gene (phaC). The insertion of a self-cleaving module, a modified sortase A (SrtA) from Staphylococcusaureus and its five amino acid recognition sequence between the synthase and the target protein led to a simple protein production and purification method. The generation of hybrid genes encoding tripartite PhaC-SrtA-Target fusion proteins, enabled immobilization of proteins of interest to the surface of PHA beads in vivo. After simple cell lysis and isolation of the PHA beads, the target proteins could be selectively and efficiently released form the beads by activating the sortase with CaCl2 and triglycine. Up to 6 mg/l of soluble proteins at a purity of ~98 % could be isolated in one step with no optimization. This process was used to produce and isolate three proteins: Green fluorescent protein, maltose binding protein and the Mycobacteriumtuberculosis vaccine candidate Rv1626. We have developed a new technique for easy production and purification of recombinant proteins. This technique is capable of producing and purifying high yields of proteins suitable for research application in less than 2 days. No costly or specialized protein chromatography equipment, resins, reagents or expertise are required. The online version of this article (doi:10.1186/s12934-015-0385-3) contains supplementary material, which is available to authorized users.