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

Takanori Otomo, Kenta Teruya, Koichi Uegaki, Toshio Yamazaki, Yoshimasa Kyogoku

Research output: Contribution to journalArticlepeer-review

79 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)105-114
Number of pages10
JournalJournal of biomolecular NMR
Volume14
Issue number2
DOIs
Publication statusPublished - 1999
Externally publishedYes

Keywords

  • Intein
  • Larger protein
  • Protein splicing
  • Segmental isotope labeling

ASJC Scopus subject areas

  • Biochemistry
  • Spectroscopy

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