Chemical synthesis and spontaneous folding of a multidomain protein: Anticoagulant microprotein S

Because of recent high-yield native ligation techniques, chemical synthesis of larger multidomain bioactive proteins is rapidly coming within reach. H ere we describe the total chemical synthesis of a designed "microprotein S, " comprising the gamma -carboxyglutamic acid-rich module, the thrombin-sens itive module, and the first epidermal growth factor-like module of human pl asma protein S (residues 1-116). Synthetic microprotein S expressed anticoa gulant cofactor activity for activated protein C in the down-regulation of blood coagulation, and the anticoagulant activity of microprotein S was not neutralized by C4b-binding protein, a natural inhibitor of native protein S in plasma. The correct folding of this complex multidomain protein was en hanced compared with individual modules because the gamma -carboxyglutamic acid-rich module and the thrombin-sensitive module markedly facilitated cor rect folding of the first epidermal growth factor-like module compared with folding of the first epidermal growth factor-like module alone. These resu lts demonstrate that total chemical synthesis of proteins offers an effecti ve way to generate multidomain biologically active proteins.