Otomo T, Teruya K, Uegaki K, Yamazaki T, Kyogoku Y
Institute for Protein Research, Osaka University, Japan.
J Biomol NMR. 1999 Jun;14(2):105-14. doi: 10.1023/a:1008308128050.
A new isotope labeling technique for peptide segments in a protein sample was recently established using the protein splicing element intein [Yamazaki et al. (1998) J. Am. Chem. Soc., 120, 5591-5592]. This method makes it possible to observe signals of a selected amino (N-) or carboxyl (C-) terminal region along a peptide chain. However, there is a problem with the yield of the segmentally labeled protein. In this paper, we report an increase in the yield of the protein that enables the production of sufficient amounts of segmentally 13C/15N-labeled protein samples. This was achieved by improvement of the expression level of the N-terminal fragment in cells and the efficiency of refolding into the active splicing conformation. The N-terminal fragment was expressed as a fused protein with the cellulose binding domain at its N-terminus, which was expressed as an insoluble peptide in cells and the expression level was increased. Incubation with 2.5 M urea and 50% glycerol increased the efficiency of the refolding greatly, thereby raising the final yields of the ligated proteins. The feasibility of application of the method to a high-molecular-weight protein was demonstrated by the results for a maltose binding protein consisting of 370 amino acids. All four examined joints in the maltose binding protein were successfully ligated to produce segmentally labeled protein samples.
最近，利用蛋白质剪接元件内含肽建立了一种用于蛋白质样品中肽段的新同位素标记技术[Yamazaki等人（1998年），《美国化学会志》，120，5591 - 5592]。该方法能够沿着肽链观察选定的氨基（N -）或羧基（C -）末端区域的信号。然而，片段标记蛋白质的产量存在问题。在本文中，我们报告了蛋白质产量的提高，这使得能够生产出足够量的片段13C/15N标记蛋白质样品。这是通过提高细胞中N末端片段的表达水平以及重折叠成活性剪接构象的效率来实现的。N末端片段作为在其N末端与纤维素结合结构域融合的蛋白质进行表达，该结构域在细胞中以不溶性肽的形式表达且表达水平得到提高。用2.5 M尿素和50%甘油孵育大大提高了重折叠效率，从而提高了连接蛋白质的最终产量。由370个氨基酸组成的麦芽糖结合蛋白的结果证明了该方法应用于高分子量蛋白质的可行性。麦芽糖结合蛋白中所有四个检测的接头都成功连接，产生了片段标记蛋白质样品。
