Chemical ligation of folded recombinant proteins: segmental isotopic labeling of domains for NMR studies.
A convenient in vitro chemical ligation strategy has been developed that allows folded recombinant proteins to be joined together. This strategy permits segmental, selective isotopic labeling of the product. The src homology type 3 and 2 domains (SH3 and SH2) of Abelson protein tyrosine kinase, which constitute the regulatory apparatus of the protein, were individually prepared in reactive forms that can be ligated together under normal protein-folding conditions to form a normal peptide bond at the ligation junction. This strategy was used to prepare NMR sample quantities of the Abelson protein tyrosine kinase-SH(32) domain pair, in which only one of the domains was labeled with 15N. Mass spectrometry and NMR analyses were used to confirm the structure of the ligated protein, which was also shown to have appropriate ligand-binding properties. The ability to prepare recombinant proteins with selectively labeled segments having a single-site mutation, by using a combination of expression of fusion proteins and chemical ligation in vitro, will increase the size limits for protein structural determination in solution with NMR methods. In vitro chemical ligation of expressed protein domains will also provide a combinatorial approach to the synthesis of linked protein domains.
Xu
R
R
Laboratory of Physical Biochemistry, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.
Ayers
B
B
Cowburn
D
D
Muir
T W
TW
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009587:Nitrogen Isotopes; D016374:Oncogene Proteins v-abl / Q000737:chemistry; D015202:Protein Engineering; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D018909:src Homology Domains / Q000235:genetics
D009587:Nitrogen Isotopes; D016374:Oncogene Proteins v-abl; D011993:Recombinant Fusion Proteins