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

Abstract

Hirudin-1 is a highly selective inhibitor of thrombin secreted by salivary glands of the medicinal leech Hirudo medicinalis. This direct anticoagulant is used for the treatment and prevention of disorders in blood coagulation system. Apart from the existing recombinant analog of hirudin-1 (63-desulfatohirudin-1, desirudin) its modified analogs possessing higher activity and stability are of medical value. In this study artificial genes of hirudin and two its analogs (hirudin-1, [Leu1, Thr2]-hirudin-1 and [Leu1, Thr2]-hirudin-1/3) were synthesized and cloned in an expression vector pTWIN1 in frame with the gene of mini-intein SspDnaB 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 analogs were developed. The scheme includes the following stages: isolation of the fusion protein after the desintegration of the cell biomass, refolding of the target peptide within the fusion protein, pH-inducible cleavage of the fusion protein, and chromatographic purification of the target product. Antithrombotic activity of the obtained peptides was determined by a standard amidolytic assay. The developed methods for the production of 63-desulfatohirudin-1, [Leu1, Thr2]-desulfatohirudin-1 [Leu1, Thr2]-desulfatohirudin-1/3 allowed to obtain these peptides with high yields (14, 25 and 24 mg per liter of cell culture respectively) and high activity (13423, 33333 and 19802 ATU/mg respectively).

Screening of the Promising Direct Thrombin Inhibitors from Haematophagous Organisms. Part I: Recombinant Analogues and Their Antithrombotic Activity In Vitro

“…The affinity of each peptide increased, in a non-statistically significant manner, after incubation with thrombin, which is typical for fast, tight-binding inhibitors ( Figure S5 ). The values for hirudin-1 and anophelin were similar to those determined previously [ 36 ]. The values calculated for haemadin and variegin coincide with those estimated by other authors [ 37 ].…”

“…Hirudin-1isthe first-known and most well-studied natural bivalent thrombin inhibitor withinhibitions constants of about 200 fM.The structure of hirudin-1 includes a [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 ]-core domain that binds to the thrombin active site and unstructured [ 55 , 56 , 57 , 58 , …”

“…Anophelin is a cysteine-less bivalent thrombin inhibitor with a Ki value of about 30–100 pM, but with a unique inhibition mechanism: it binds to the active center and exosite-1, but in a reverse orientation [ 33 , 64 ]. The [ 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 ]-fragment block of the thrombin exosite-1 and the [ 50 , 51 , 52 , 53 ]-tetrapeptide interact with its active site. Although a 31 aa N-terminal fragment of anophelindoes not possess the antithrombin activity, due to its negative charge it can promote the correct formation of the complex with thrombin.…”

“…Thromborel-S, Pathromtin SL, and Test Thrombin reagents from Siemens Healthcare Diagnostic (Erlangen, Germany) were used in coagulation tests with human plasma. Recombinant hirudin-1 and anophelin were obtained according to the protocol published previously [ 36 , 37 ]. Other reagents used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Panreac (Barcelona, Spain).…”

“…In our previous papers, we have reported the production of the recombinant hirudin-1 and anophelin, an anticoagulant from the malaria mosquito Anopheles albimanus [ 36 , 37 ]. According to the published data, solid-phase peptide synthesis is still the only effective method to produce variegin [ 38 ].…”