Prevention of NH2-terminal acetylation of proteins synthesized in cell-free systems

Abstract

The NH2 terminus of ovalbumin is acetylated in cell-free protein-synthesizing systems as it is in vivo. The acetyl group is derived from acetyl-CoA and it is incorporated during translation. Acetylation can be prevented by metabolizing the available acetyl-CoA to citrate with the addition of citrate synthase and oxalacetate to the translation system. The NH2 terminus of ovalbumin synthesized under these conditions can be sequenced by automated Edman degradation. This procedure has also been applied to the sequencing of Pr 76gag, the viral core protein precursor synthesized from 35 S Rous sarcoma virus RNA.