Expression and Affinity Purification of Small Molecule Functional Peptide of Hirudin in E. coli

Hirudin has been well-characterized to be the strongest thrombin inhibitor with no affinity for other peptidases. Hirudin from natural sources is inefficient due to its low abundance in medicinal leech (Hirudo medicinalis). Though recombinant hirudin has been made by many laboratories, technical difficulties have limited recombinant hirudin from being used clinically. One of the difficulties is the purification of recombinat hirudin which is usually tedious and inefficient. Secondly, it has been difficult to generate a small molecule derivative of hirudin that maintains or has increased antithrombotic activity so that wide clinical application may be possible. To express recombinant fusion proteins of the GST affinity tag with the hirudin functional peptide in Escherichia coli, the DNA sequence of the functional domain of hirudin peptide was synthesized, and subsequently inserted into the E.coli expression vector pGEX-6p-l. The resulting plasmid, termed pGEX-6p-1-HIR, was transformed into E.coli strain DH5alpha. Expression of the recombinant protein GST-HIR was then induced by IPTG. The cells were lysed by sonication, and the recombinant proteins were purified by GST affinity chromatography. The affinity purified recombinant proteins were then incubated with PreScission Protease for 4 hours to cleave the GST-HIR fusion proein. The antithrombotic activity of the hirudin functional peptide was determined by direct titration with thrombin. The small molecule functional peptides of hirudin we finally generated were 26 amino acid residues long, and the antithrombotic activity of the hirudin functional peptide reached as high as 653 ATU/mg.