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.
Cuicui Huang, Xuerui Zhang, Jia Qu, Ping Zhang, Shuhua Tan
Endothelial nitric oxide synthase polymorphism G298T in association with oxidative DNA damage in coronary atherosclerosis
Content Type Journal Article
Category Research Note
RAJESH G. KUMAR, Department of Genetics, Osmania Univers ... more
A micropropagation protocol was developed for multiplication of seedless lemon (Citrus limon L. cv. Kaghzi Kalan) using nodal explants. The maximum shoot regeneration was observed on low level of BAP (0.1 mg l−1) or kinetin (0.5 mg l−1). BA was recorded to be better than kinetin in terms of ... more
The objective of this study was to evaluate the activity, kinetic behavior, and thermal inactivation kinetics of peroxidase (POD) in aqueous extracts from two kinds of milk ripe stage corn, sweet corn and waxy corn. Optimum activities using guaiacol as the hydrogen donor were obtained for s ... more