Morganti Ligia, Chura-Chambi Rosa Maria
Centro de Biotecnologia, Instituto de Pesquisas Energéticas e Nucleares, IPEN-CNEN/SP, São Paulo, SP, Brazil.
Methods Mol Biol. 2023;2617:177-187. doi: 10.1007/978-1-0716-2930-7_12.
The expression of recombinant proteins as insoluble inclusion bodies (IB) has the advantage to separate insoluble aggregates from soluble bacterial molecules, thus obtaining proteins with a high degree of purity. Even aggregated, the proteins in IB often present native-like secondary and tertiary structures, which can be maintained as long as solubilization is carried out in non-denaturing condition. High pressure solubilizes IB by weakening hydrophobic interactions, while alkaline pH solubilizes aggregates by electrostatic repulsion. The combination of high pressure and alkaline pH is effective for IB solubilization at a mild, non-denaturing condition, which is useful for subsequent refolding. Here, we describe the expression of recombinant proteins in Escherichia coli using a rich medium to obtain high expression levels, bacterial lysis, and washing of the IB to obtain products of high purity, and, finally, the solubilization and high yield of refolded proteins using high pressure and alkaline pH.
将重组蛋白表达为不溶性包涵体（IB）具有从可溶性细菌分子中分离不溶性聚集体的优势，从而获得高纯度的蛋白质。即使处于聚集状态，IB中的蛋白质通常也呈现类似天然的二级和三级结构，只要在非变性条件下进行溶解，这些结构就能得以维持。高压通过削弱疏水相互作用使IB溶解，而碱性pH值则通过静电排斥使聚集体溶解。高压和碱性pH值的组合在温和的非变性条件下对IB溶解有效，这对后续的重折叠很有用。在此，我们描述了在大肠杆菌中使用丰富培养基表达重组蛋白以获得高表达水平、细菌裂解以及洗涤IB以获得高纯度产物，最后使用高压和碱性pH值溶解并高产率重折叠蛋白质的过程。
