Cloning and Expression in Escherichia coli of a Synthetic DNA for Hirudin,the Blood Coagulation Inhibitor in the Leech

Abstract

A 235-bp DNA coding for the leech blood coagulation inhibitor, hirudin, was chemically synthesized. The synthesis involved preparation of seven long oligodeoxyribonucleotide pairs which were assembled and cloned using a rapid and simple procedure. More than half of the transformed Escherichia coli cells expressed a biosynthetic polypeptide having biological properties which were very similar to authentic hirudin from the leech Hirudo medicinalis. To achieve efficient expression, we fused the hirudin DNA to a truncated C1 repressor gene of bacteriophage λ to create a hybrid protein. An additional methionine at the fusion point allowed the active hirudin to be cleaved off by cyanogen bromide.