Improved segmental isotope labeling of proteins and application to a larger protein

A new isotope labeling technique for peptide segments in a protein sample w as recently established using the protein splicing element intein [Yamazaki et al. (1998) J. Am. Chem. Soc., 120, 5591-5592]. This method makes it pos sible 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 o f 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 C-13/N-15-labeled protein samples. This was achieved by impr ovement of the expression level of the N-terminal fragment in cells and the efficiency of refolding into the active splicing conformation. The N-termi nal fragment was expressed as a fused protein with the cellulose binding do main at its N-terminus, which was expressed as an insoluble peptide in cell s and the expression level was increased. Incubation with 2.5 M urea and 50 % glycerol increased the efficiency of the refolding greatly, thereby raisi ng the final yields of the ligated proteins. The feasibility of application of the method to a high-molecular-weight protein was demonstrated by the r esults for a maltose binding protein consisting of 370 amino acids. All fou r examined joints in the maltose binding protein were successfully ligated to produce segmentally labeled protein samples.