Hirudin variant III (HV3) is potentially useful in the prevention and treatment of cataracts. To prepare sufficient amounts of rHV3 for further preclinical studies, we developed an effective process for robust preparative-scale extracellular production of rHV3 in Escherichia coli. In a 7-l bioreactor, under the optimal fed-batch fermentation conditions, rHV3 was excreted into the culture supernatant and yielded up to 915 mg l(-1). Then, a four-step purification procedure was applied to the product, which included ultrafiltration, hydrophobic chromatography, anion-exchange chromatography, and preparative reversed-phase fast protein liquid chromatography (FPLC). The overall maximum recovery attained was 56 %, the purity reached at least 99 % as evaluated by HPLC analysis, the molecular weight was determined to be 7,011.10 Da by matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF/MS) analysis, and the pI was 4.46 as analyzed by isoelectric focusing. The N- and C-terminal sequence analysis confirmed the product homogeneity. The final product contained at most 10 pg of residual DNA per dose (0.2 mg) of rHV3 by high-sensitivity hybridization assay and at most 3 EU endotoxin protein/mg by limulus amebocyte lysate assay. Taken together, the rHV3 produced in multigram quantities in E. coli by this bioprocess meets the regulatory criteria for biopharmaceuticals and can be used as a drug candidate for preclinical studies.

Hirudin can be used as an oral anticoagulant and antithrombotic agent. The hirudin variant III gene, derived from the medicinal leech, Hirudo medicinalis, was fused to SP310mut2 signal sequence and expressed by a nisin-controlled gene expression system in Lactococcus lactis which was then grown in a 7 l fermenter. After induction with 8 ng nisin ml(-1), the product was secreted into the culture medium and accumulated up to ~2.7 mg l(-1). MALDI-TOF/MS and anticoagulant activity analyses on the purified product confirmed its authenticity. This is the first demonstration that hirudin can be extracellularly secreted and correctly processed in L. lactis.