Facultad de Ciencias Quimicas, Universidad Autonoma de Nuevo Leon, San Nicolas de los Garza, NL, Mexico.
Institute for Society & Genetics, Department of Microbiology, Immunology & Molecular Genetics, University of California, Los Angeles, CA, USA.
Methods Mol Biol. 2021;2178:329-344. doi: 10.1007/978-1-0716-0775-6_22.
The bacterium Escherichia coli is still considered the first option as a microbial cell factory for recombinant protein production, and affinity chromatography is by far the preferred technique for initial purification after protein expression and cell lysis. In this chapter, we describe the methodology to express and purify recombinant proteins in E. coli tagged with the first two metal-binding proteins proposed as fusion partners. They are the small metal-binding protein SmbP and a mutant of the copper resistance protein CusF3H+. There are several advantages of using them as protein tags: they prevent the formation of inclusion bodies by increasing solubility of the target proteins, they enable purification by immobilized metal-affinity chromatography using Ni(II) ions with high purity, and because of their low molecular weights, excellent final yields are obtained for the target proteins after cleavage and removal of the protein tag. Here we also describe the protocol for the production of proteins in the periplasm of E. coli tagged with two SmbP variants that include the PelB or the TorA signal sequences for transport via the Sec or the Tat pathway, respectively. Based on these methods, we consider CusF3H+ and SmbP excellent alternatives as fusion proteins for the production of recombinant proteins in E. coli.
大肠杆菌仍然被认为是用于重组蛋白生产的微生物细胞工厂的首选，亲和层析是在蛋白表达和细胞裂解后进行初始纯化的首选技术。在本章中，我们描述了使用两种金属结合蛋白（SmbP 和一种铜抗性蛋白 CusF3H+ 的突变体）作为融合伴侣表达和纯化标记的重组蛋白的方法。使用它们作为蛋白标签有几个优点：它们通过增加目标蛋白的溶解性来防止包涵体的形成，它们可以使用 Ni(II)离子通过固定化金属亲和层析进行纯化，并且由于它们的分子量低，在切割和去除蛋白标签后，目标蛋白可以获得优异的最终产量。在这里，我们还描述了使用两种 SmbP 变体在大肠杆菌周质中标记蛋白的方案，这些变体分别包含 PelB 或 TorA 信号序列，用于通过 Sec 或 Tat 途径进行运输。基于这些方法，我们认为 CusF3H+ 和 SmbP 是大肠杆菌中生产重组蛋白的融合蛋白的优秀替代品。
