POSITIONAL CONTROL OF SELENIUM INSERTION IN POLYPEPTIDES FOR X-RAY CRYSTALLOGRAPHY

摘要

The invention provides methods for determining the structure of a polypeptide by a) transfecting a cell with (i) a first nucleic acid encoding the polypeptide, wherein at least one, specific codon of mRNA transcribed from the first nucleic acid is replaced by the codon UGA, and (ii) a second nucleic acid, operably linked to the first nucleic acid, that directs the translation of the UGA codon as selenocysteine only when the cell can obtain selenium from the medium in which it is grown; b) growing the cell in selenium-containing growth medium under conditions in which the cell incorporates at least one selenocysteine residue into the polypeptide at a specific location; c) isolating the polypeptide from the cell or the growth medium; d) forming a crystal of the polypeptide; and e) performing X-ray crystallography on the crystal, wherein the selenocysteine residue is used to determine the structure of the polypeptide.