Biotechnological production of recombinant analogues of hirudin-1 from Hirudo medicinalis

• Published in: Russian journal of bioorganic chemistry Vol. 38; no. 2; pp. 142 - 151
• Main Authors: Kostromina, M. A., Esipov, R. S., Miroshnikov, A. I.
• Format: Journal Article
• Language: English
• Published: Dordrecht SP MAIK Nauka/Interperiodica 01.03.2012
• Subjects: Anticoagulants; Biochemistry; Biomedical and Life Sciences; Biomedicine; Bioorganic Chemistry; Blood coagulation; Cell culture; Escherichia coli; Expression vectors; Fusion protein; Hirudinea; Hirudo medicinalis; Life Sciences; Organic Chemistry; protein purification; Renaturation; Salivary gland; Synechocystis; Thrombin; 63-desulfatohirudin-1; antithrombotic activity; intein
• ISSN: 1068-1620; 1608-330X
• DOI: 10.1134/S1068162012020057

Hirudin-1 is a highly selective inhibitor of thrombin secreted by the salivary glands of the medicinal leech Hirudo medicinalis . This direct anticoagulant is used for the treatment and prevention of disorders in the blood coagulation system. Apart from the existing recombinant analogue of hirudin-1 (63-desulfato-hirudin-1) its modified analogues possessing higher activity and stability are of medical value. In this study artificial genes of hirudin-1 and two its analogues ([Leu 1 , Thr 2 ]-hirudin-1 and [Leu 1 , Thr 2 ]-hirudin-1/3) were synthesized and cloned in an expression vector pTWIN1 in frame with the gene of mini-intein DnaB from Synechocystis sp. Producing strains of the corresponding fusion proteins were constructed using E. coli strain ER2566. Biotechnological schemes for the production of 63-desulfatohirudin-1 and its analogues were developed. The scheme includes the following stages: isolation of the fusion protein, renaturation of the target protein incorporated into the fusion protein, pH-inducible cleavage of the fusion protein, and chromatographic purification of the target product. Antithrombotic activity of the peptides obtained was determined by a standard amidolytic assay. The developed methods for the production of 63-desulfatohirudin-1, [Leu 1 , Thr 2 ]-63-desulfatohirudin-1 and [Leu 1 , Thr 2 ]-63-desulfatohirudin-1/3 allowed us to obtain these peptides with high yields (14, 25 and 24 mg per 1 L of cell culture, respectively) and high activity (13423, 33333 and 19802 ATU/mg, respectively).