Improved segmental isotope labeling of proteins and application to a larger protein.
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.
Otomo
T
T
Institute for Protein Research, Osaka University, Japan.
Teruya
K
K
Uegaki
K
K
Yamazaki
T
T
Kyogoku
Y
Y
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D002247:Carbon Isotopes; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005990:Glycerol; D007202:Indicators and Reagents; D007553:Isotope Labeling / Q000379:methods; D058619:Maltose-Binding Proteins; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry; D019154:Protein Splicing; D017433:Protein Structure, Secondary; D019711:Pyrococcus / Q000201:enzymology; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000235:genetics; D014508:Urea
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002247:Carbon Isotopes; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D007202:Indicators and Reagents; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D009587:Nitrogen Isotopes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D014508:Urea; D012264:Ribonucleotide Reductases; D005990:Glycerol