• Biomolecules & Therapeutics
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• v.1 no.2
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• pp.166-170
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• 1993

Hirudin is a potent inhibitor of thrombin, which was originally obtained from the medicinal leech (Hirudo medicinalis) Now it is being produced through the recombinant technology on a large scale. Recombinant hirudin has been assayed for the anticoagulant activity by the measurement of clotting time and the inhibition of thrombin actvity using a chromogenic substrate. The assay range of partial thromboplastin time and thrombin time is within $0.2{\sim}1.0 {\mu}g/mι.$ Thrombin time is more sensitive to the measurement of clot. Ex vivo study showed the level of hirudin in rat plasma was highest in 10 min and then it was eliminated slowly. The half-life of r-hirudin was 80~110 min depending on the assay methods. Intraveneous injection of russel viper venom was used for thrombus induction combined with vents cava ligation. Inhibition of venous thrombosis was observed with i.v. hirudin. It was dependent on the concentration of hirudin.

• KSBB Journal
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• v.13 no.4
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• pp.456-460
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• 1998

Fed-batch fermentations were carried out in order to improve the efficiency of hirudin production by recombinant Saccharomyces cerevisiae. A fed-batch fermentation done with the optimized semi-synthetic medium resulted in a maximum hirudin concentration of 342mg/$\ell$ by keeping a galactose concentrations between 10 and 30g/$\ell$ which corresponded to a 11.4-fold increase in hirudin concentration compared with the simple bach fermentation done with the same medium. Comparison of the chromatographic pattern of proteins in the growth medium clearly showed that the use of the semi-synthetic medium is more advantageous for separation of hirudin than the case o fusing the complex medium. Continuous cell recycle fermentation done at dilution rate of 0.1h-1 and an inlet galactose concentration of 100g/$\ell$ yielded a maximum hirudin productivity of 19.1mg hirudin/$\ell$$.$h.

• Biotechnology and Bioprocess Engineering:BBE
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• v.8 no.3
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• pp.183-186
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• 2003

The artificial gene coding for anticoagulant hirudin was placed under the control of the GAL 10 promoter and expressed in the galactokinase-deficient strain (Δgal1) of Saccharomyces cerevisiae, which uses galactose only as a gratuitous inducer in order to avoid its consumption. For efficient production of recombinant hirudin, a carbon source other than galactose should be provided in the medium to support growth of the Δgal1 strain. Here we demonstrate the successful use of glucose in the fed-batch fermentation of the Δgal1 strain to achieve efficient production of recombinant hirudin, with a yield of up to 400 mg hirudin/L.

• Journal of Microbiology and Biotechnology
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• v.1 no.4
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• pp.266-273
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• 1991

Hirudin, a 65-amino acid protein isolated from the salivary gland of the bloodsucking leech, Hirudo medicinalis, is a potent thrombin-specific inhibitor and blocks the thrombin-mediated conversion of fibrinogen to fibrin in clot formation. We have studied the gene expression and secretion of hirudin in yeast. Saccharomyces cerevisiae. A gene coding for hirudin was synthesized based on the amino acid sequence and cloned into a yeast expression vector $YEG{\alpha}-1$ containing the ${\alpha}-mating$ factor pre-pro leader sequence and galactose-inducible promoter, GALl0. Recombinant S. cerevisiae was found to secrete biologically active hirudin into the extracellular medium. The secreted recombinant hirudin was recovered from the culture medium and purified with ultrafiltration and reverse phase high performance liquid chromatography. Approximately 1 mg of hirudin per liter was produced under suboptimal culture conditions and brought to about 90% purity in two steps of purification.

• Journal of Microbiology and Biotechnology
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• v.9 no.2
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• pp.179-183
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• 1999

A recombinant form of hirudin, a potent thrombin-specific inhibitor derived from the bloodsucking leech, was expressed as a secretory product in Saccharomyces cerevisiae under the control of GALl0 promoter and the mating factor $\alpha$pre-pro leader sequence. In an attempt to produce recombinant hirudin (r-Hir) of therapeutic purity in large quantities, the fed-batch fermentation was carried out by using this recombinant yeast, and subsequently downstream processing was developed with the preparative-scale column chromatography systems. About 234 mg/l of biologically active r-Hir was produced as a secretory product by the fed-batch fermentation strategy developed for an efficient downstream processing. Using a two-step chromatography process (an anion exchange chromatography followed by the reverse phase HPLC), the r-Hir was purified to>98% with an overall recovery yield of 84%. According to the N-terminal amino acid sequencing, the purified r-Hir was found to have the predicted N-terminal amino acid sequence. The biological activity of the purified r-Hir to inhibit thrombin was also identical to that of the commercial hirudin.

• KSBB Journal
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• v.9 no.1
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• pp.8-15
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• 1994

The research has been carried out to optimize a recombinant S. cerevisine fermentation process for the production of an anticoagulant hirudin. The structural gene coding for hirudin was combined with the GAL10 promoter for controlled expression, the MFal signal sequence for hirudin secretion, and the GAL7 terminator for transcriptional termination. Growth medium composition and environmental conditions were optimized for maximizing cell growth and final hirudin concentration. The optimized conditions included yeast extract 40g/$\ell$, casamino acid 5g/$\ell$, g1ucose 20g/$\ell$, galactose 30g/$\ell$, DO 50% and temperature $30^{\circ}C$. These conditions yielded the specific cell growth rate of $0.13hr^{-1}$, the final cell density of 30g cell/$\ell$ and the final hirudin concentration of 64mg/$\ell$ in the batch fermentation with a 2.5$\ell$ jar fermentor.

• YAKHAK HOEJI
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• v.41 no.1
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• pp.74-80
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• 1997

A polygonal antibody against recombinant hirudin was raised for the development of a ELISA in biological fluids. Recombinant hirudin was conjugated to maleimide activated carrie r protein, KLH and injected to a rabbit. The third booster collection of antiserum was used as primary antibody for the ELISA. The titer for the detection antibody was determined. The direct ELISA could determine the concentration of hirudin in the range of ~10ng/ml. Affinity pulified IgG was obtained and conjugated to horseradish peroxidase. Purified IgG and IgG-HRP could be used as capture and detection antibody, respectively. Although sandwich ELISA would not give the satisfactory results. it could apply for the detection of hirudin level in the range of ~20 ${\mu}$g/ml.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.99-102
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• 2001

Recombinant Saccharomyces cerevisiae strains harboring various copy numbers of hirudin gene were developed to study dependency of hirudin expression level on its gene copy number. A linear relationship between the copy number of hirudin expression cassette and hirudin expression level was observed up to 10 copies. A double <5-integration vector truncated wi 디 1 the unnecessary bacterial genes before yeast transformation showed a four-fold increase in transformation efficiency and a 1.3-fold enhancement in hirudin expression level compared with a single <5 system. Gratuitous hirudin expression strain was developed by disrupting the GALl gene of S. cerevisiae. Glucose that was fed in a limited manner effectively supported cell growth and hi겨din expression by the gratuitous strain. Effects of methanol concentrations on hirudin production in recombinant Hansenula polymorpha were investigated in continuous and fed-batch cultures. At a steady-state of continuous culture, an optimum methanol concentration of 1.7 g/L was determined at a dilution rate of 0.18 $h^{-1}$ with 1.8 mg/L ${\cdot}$ h hirudin productivity.

• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.1
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• pp.1-6
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• 2000

For the rapid selection of higher recombinant hirudin producing strain in a methylotrophic yeast Hansenula polymorpha, a multiple gene integration and dose-dependent selection vector, based on a telomere-associated ARS and a bacterial aminoglycoside 3-phosphotransferase (aph) gene, was adopted. Two hirudin expression cassettes (HV1 and HV2) were constructed using the MOX promoter of H. polymorpha and the mating factor $\alpha$ secretion signal of S. cerevisiae. Multiple integrants of a transforming vector containing hirudin expression cassettes were easily selected by using an antibiotic, G418. Hirudin expression level and integrated plasmid copy number of the tested transformants increased with increasing the concentration of G418 used for selection. The expression level of HV1 was consistently higher than that of HV2 under the similar conditions, suggesting that the gene context might be quite important for the high-level gene expression in H. polymorpha. The highest hirudin producing strain selected in this study produced over 96 mg/L of biologically active hirudin in a 500-mL flask and 165 mg/L in a 5-L fermentor.

• Biotechnology and Bioprocess Engineering:BBE
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• v.8 no.5
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• pp.303-305
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• 2003

Scale-up of hirudin production from Saccharomyces cerevisiae from bench-scale to pilot-scale was carried out based on constant volumetric oxygen transfer coefficient (K$\sub$L/a). Fed-batch mode of cultivation using step-wise feeding strategy of galactose was employed for the production of hirudin in a 30-L and a 300-L pilot-scale fermentor. The final hirudin concentrations were achieved 390 mg/L and 286.1 mg/L, and the volumetric productivities were 80.4% and 90.7% with the 30-L and 300-L fermentors, respectively, compared to the productivity of the 5-L bench-scale fermentor.