Methods for isotopically labeling biomolecules using mammalian cell-free extracts

An in vitro method for producing heavy isotope-labeled protein, the method comprising

• combining a mammalian cell-free extract with at least one DNA, at least one RNA polymerase, at least one accessory expression protein, an ATP regeneration system, and a plurality of heavy isotope-labeled amino acids and/or heavy isotope-labeled tRNAs to form an in vitro transcription/translation system without supplementing with exogenous tRNA and without supplementing with light amino acids, and
• incubating the in vitro transcription/translation system under conditions to result in production of at least one heavy isotope-labeled functional protein from the at least one DNA.

An in vitro method for producing heavy isotope-labeled protein, the method comprising

• combining a mammalian cell-free extract with at least one DNA, at least one RNA polymerase, an ATP regeneration system, and a plurality of non-heavy isotope-labeled amino acids and/or non-heavy isotope-labeled tRNAs,
• thereafter adding a plurality of corresponding heavy isotope-labeled amino acids and/or heavy isotope-labeled tRNAs in a concentration sufficient to replace the non-heavy isotope-labeled amino acids and/or non-heavy isotope labeled tRNAs to form an in vitro transcription/translation system, and
• incubating the in vitro transcription/translation system under conditions to result in production of at least one heavy isotope-labeled functional protein from the at least one DNA.