Mills K V, Lew B M, Jiang S, Paulus H
Boston Biomedical Research Institute, 20 Staniford Street, Boston, MA 02114, USA.
Proc Natl Acad Sci U S A. 1998 Mar 31;95(7):3543-8. doi: 10.1073/pnas.95.7.3543.
Protein splicing involves the self-catalyzed excision of protein splicing elements, or inteins, from flanking polypeptide sequences, or exteins, leading to the formation of new proteins in which the exteins are linked directly by a peptide bond. To study the enzymology of this interesting process we have expressed and purified N- and C-terminal segments of the Mycobacterium tuberculosis RecA intein, each approximately 100 amino acids long, fused to appropriate exteins. These fragments were reconstituted into a functional protein splicing element by renaturation from 6 M urea. When renaturation was carried out in the absence of thiols, the reconstituted splicing element accumulated as an inactive disulfide-linked complex of the two intein fragments, which could be induced to undergo protein splicing by reduction of the disulfide bond. This provided a useful tool for separately investigating the requirements for the reconstitution of the intein fragments to yield a functional protein splicing element and for the protein splicing process per se. For example, the pH dependence of these processes was quite different, with reconstitution being most efficient at pH 8.5 and splicing most rapid at pH 7.0. The availability of such an in vitro protein splicing system opens the way for the exploration of intein structure and the unusual enzymology of protein splicing. In addition, this trans-splicing system is a potential protein ligase that can link any two polypeptides fused to the N- and C-terminal intein segments.
蛋白质剪接涉及从侧翼多肽序列（即外显肽）中自我催化切除蛋白质剪接元件（即内含肽），从而形成新的蛋白质，其中外显肽通过肽键直接相连。为了研究这一有趣过程的酶学，我们表达并纯化了结核分枝杆菌RecA内含肽的N端和C端片段，每个片段约100个氨基酸长，并与合适的外显肽融合。这些片段通过从6 M尿素中复性而重新组装成功能性蛋白质剪接元件。当在没有硫醇的情况下进行复性时，重新组装的剪接元件以两个内含肽片段的无活性二硫键连接复合物形式积累，通过还原二硫键可诱导其进行蛋白质剪接。这为分别研究内含肽片段重新组装以产生功能性蛋白质剪接元件的要求以及蛋白质剪接过程本身提供了一个有用的工具。例如，这些过程对pH的依赖性差异很大，复性在pH 8.5时效率最高，剪接在pH 7.0时最快。这种体外蛋白质剪接系统的可用性为探索内含肽结构和蛋白质剪接的异常酶学开辟了道路。此外，这种反式剪接系统是一种潜在的蛋白质连接酶，可连接融合到N端和C端内含肽片段的任何两个多肽。
