Production of the HV1 variant of hirudin by recombinant DNA methodology

Poët, P de Taxis du; Scacheri, Emanuela; Benatti, Luca; Nitti, Gianpaolo; Valsasina, Barbara; Sarmientos, Paolo

doi:10.1097/00001721-199102000-00018

Cited by 7 publications

(2 citation statements)

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“…High levels of HM2 were achieved using an expressionlsecretion vector containing an oligonucleotide sequence coding for OmpA leader peptide [20] fused to a synthetic gene coding for HM2 [S]. The final preparation of mature HM2 was more than 95 O/ o homogeneous after purification by two ionexchange chromatographic steps, while the remaining 5 O/o was constituted by four differently degraded HM2derived peptidic chains.…”

Section: On the Postsynthetic Fate Of Recombinant Hirudinmentioning

confidence: 99%

Investigation on minor degraded derivatives of the recombinant hirudin variant HM2 from Hirudinaria manillensis isolated by isoelectric focusing in multicompartment electrolyzers

et al. 1996

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On isoelectric focusing in immobilized pH gradients (IPG) a preparation of recombinant hirudin from Hirudinaria manillensis, purified to homogeneity, was found to still contain a total of 5% minor components: three with higher pI values (pIs 4.10, 4.25 and 4.31), one with a lower pI value (pI 3.98) as compared with the main form (pI 4.03). Multicompartment electrolyzers with isoelectric membranes and micropreparative IPG gel slabs allowed the recovery of pure fractions of such minor components, which were further characterized by electrospray mass spectra, limited proteolysis, and sequence analysis. All four minor isoforms were found to be cleavage products of the parent, full-length hirudin molecule (molecular mass 6797 Da), as follows: the pI 4.31 (5032 Da) had lost sixteen amino acids from the N-terminus, the pI 4.25 (6212 Da) lacked five amino acids from the C-terminus, the pI 4.10 (2980 Da) was a cleavage product at residue Cys37, and the pI 3.98 (6610 Da) lacked the dipeptide Val-Ser at the N-terminus. Combining the extreme resolving power of IPGs with the high accuracy of mass spectra was found to be an attractive strategy in decoding post-synthetic modifications often encountered in r-DNA proteins.

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Section: On the Postsynthetic Fate Of Recombinant Hirudinmentioning

confidence: 99%

Investigation on minor degraded derivatives of the recombinant hirudin variant HM2 from Hirudinaria manillensis isolated by isoelectric focusing in multicompartment electrolyzers

et al. 1996

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“…Efficient production of recombinant HM2 was achieved using periplasmic expression in E. coli, according to a strategy previously described [27].…”

Section: Expression and Production Of Hm2mentioning

confidence: 99%

Novel hirudin variants from the leech Hirudinaria manillensis

Scacheri¹,

Nitti²,

Valsasina³

et al. 1993

European Journal of Biochemistry

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Novel hirudin variants isolated from the leech Hirudinaria manillensis, a leech more specialized for mammalian parasitism, are described. Isolation of antithrombin polypeptides was performed by ion-exchange chromatographies followed by an affinity chromatography step on immobilized thrombin. The major active component, antithrombin polypeptide peak 2 (HM2), and a second polypeptide, named HM1, were purified to homogeneity and their complete amino acid sequences were determined. The protein structure of the two hirudin variants include 64 amino acids with 6 cysteine residues at highly conserved positions. Comparison of the amino acid sequences of HM1 and HM2 with other known hirudins shows differences mainly in the central part and in the C-terminal region of the polypeptides. Particularly significant is the lack of a sulfated tyrosine residue in the Cterminal portion of the molecule which is replaced by aspartic acid.Polymerase chain reaction cloning techniques were used to isolate and characterize the cDNAs and determine the genomic structures of these hirudin-like polypeptides. The cDNA clones coding for the two variants indicate the expression of pre-hirudins of 84 amino acids where the first 20 residues constitute the signal peptide required for extracellular secretion. The leader sequence appears to be highly conserved for both isoforms and shares a complete similarity with the partial hirudin variant 2 (HV2) signal peptide sequence previously reported.The HM1 and HM2 gene fragments show the presence of four exons: the first one corresponding to a 20-amino-acid signal peptide while the other three exons share the full primary structure of the antithrombin polypeptides.HM2 was also efficiently produced in recombinant Escherichia coli by expressing a periplasmic construction containing the synthetic gene.Hirudin, the most potent thrombin inhibitor known, is a small polypeptide discovered over 100 years ago in the saliva of the medicinal leech Hirudo medicinalis [l]. It was first characterized biochemically by Markwardt [2, 31 and its structure was lately elucidated by Dodt et al. [4]. It is a single-chain polypeptide composed of 65 amino acids, including 6 cysteine residues, and a molecular mass of about 7000 Da. Hirudin produces its action by binding directly to thrombinCorrespondence to E. Scacheri,

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Current status of the anticoagulant hirudin: its biotechnological production and clinical practice

Sohn

Kang

Rao

et al. 2001

Applied Microbiology and Biotechnology

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Hirudin is a potent thrombin inhibitor originally derived from the medicinal leech, Hirudo medicinalis. Owing to its high affinity and specificity for thrombin, hirudin has been intensively investigated for research and therapeutic purposes. The investigation of hirudin has contributed greatly to the understanding of the mode of action of thrombin and the clotting system. Hirudin and several hirudin analogues have also been demonstrated to have several advantages as a highly specific anticoagulant over the most widely used drug, heparin. Due to the great demand for hirudin in physicochemical and clinical studies, various recombinant systems have been developed, using bacteria, yeasts, and higher eukaryotes, to obtain the biologically active hirudin in significant quantities. After 10 years of clinical applications, two recombinant hirudins and a hirudin analogue have gained marketing approval from the United States Food and Drug Administration, for several applications. Clinical trials are currently ongoing for other treatments for thrombotic disease. As a consequence, it is conceivable that hirudin may expand its therapeutic utility over heparin in the near future.

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Production of the HV1 variant of hirudin by recombinant DNA methodology

Cited by 7 publications

References 0 publications

Investigation on minor degraded derivatives of the recombinant hirudin variant HM2 from Hirudinaria manillensis isolated by isoelectric focusing in multicompartment electrolyzers

Investigation on minor degraded derivatives of the recombinant hirudin variant HM2 from Hirudinaria manillensis isolated by isoelectric focusing in multicompartment electrolyzers

Novel hirudin variants from the leech Hirudinaria manillensis

Current status of the anticoagulant hirudin: its biotechnological production and clinical practice

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