Segmental isotopic labeling of multi-domain and fusion proteins by protein trans-splicing in vivo and in vitro.
Segmental isotopic labeling is a powerful labeling technique for reducing nuclear magnetic resonance (NMR) signal overlap, which is associated with larger proteins by incorporating stable isotopes into only one region of a protein for NMR detections. Segmental isotopic labeling can not only reduce complexities of NMR spectra but also retain possibilities to carry out sequential resonance assignments by triple-resonance NMR experiments. We described in vivo (i.e., in Escherichia coli) and in vitro protocols for segmental isotopic labeling of multi-domain and fusion proteins via protein trans-splicing (PTS) using split DnaE intein without any refolding steps or alpha-thioester modification. The advantage of PTS approach is that it can be carried out in vivo by time-delayed dual-expression system with two controllable promoters. A segmentally isotope-labeled protein can be expressed in Escherichia coli within 1 d once required vectors are constructed. The total preparation time of a segmentally labeled sample can be as short as 7-13 d depending on the protocol used.
Muona
Mikko
M
Research Program in Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, Helsinki, Finland.
Aranko
A Sesilja
AS
Raulinaitis
Vytas
V
Iwaï
Hideo
H
D001483:Base Sequence; D004258:DNA Polymerase III / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics; D005822:Genetic Vectors; D066298:In Vitro Techniques; D047668:Inteins; D007553:Isotope Labeling / Q000379:methods*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D020040:Trans-Splicing
D029968:Escherichia coli Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III