Intein-based biosynthetic incorporation of unlabeled protein tags into isotopically labeled proteins for NMR studies.
Segmental isotopic labeling of proteins using protein ligation is a recently established in vitro method for incorporating isotopes into one domain or region of a protein to reduce the complexity of NMR spectra, thereby facilitating the NMR analysis of larger proteins. Here we demonstrate that segmental isotopic labeling of proteins can be conveniently achieved in Escherichia coli using intein-based protein ligation. Our method is based on a dual expression system that allows sequential expression of two precursor fragments in media enriched with different isotopes. Using this in vivo approach, unlabeled protein tags can be incorporated into isotopically labeled target proteins to improve protein stability and solubility for study by solution NMR spectroscopy.
Züger
Sara
S
Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, Saskatchewan S7N 5C9, Canada.
Iwai
Hideo
H
D002247:Carbon Isotopes; D004926:Escherichia coli / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression; D047668:Inteins; D007553:Isotope Labeling / Q000379:methods*; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010957:Plasmids / Q000378:metabolism; D011994:Recombinant Proteins
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D019204:GTP-Binding Proteins