Extracellular secretion of anticoagulant peptide hirudin in Lactococcus lactis using SP310mut2 signal peptide

Lv, Jing; Huang, Can; Zhang, Xuerui; Tan, Shuhua

doi:10.1007/s10529-011-0740-3

Cited by 7 publications

(2 citation statements)

References 15 publications

(18 reference statements)

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“…The collected samples demonstrated single bands of ~14 kDa on the coomassie brilliant blue stained tricine-PAGE (Fig. 3 lane 2 and 3), implying the formation of homodimers as reported by previous studies (19). Purification was performed on Ni-NTA, which only has affinity for positively charged compounds.…”

Section: Expression Of Boronophenylalanine-modified Hirudin and Rhirudinsupporting

confidence: 74%

Improving the bioactivity of rHirudin with boronophenylalanine site-specific modification

Xin

Liu

Sun

2015

Molecular Medicine Reports

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To improve the bioactivity of recombinant (r)Hirudin, the orthogonal pair MjBTyrRS/tRNATyr cua (made up of the boronophenylalanine, tRNA and tRNA synthetase), was selected to incorporate boronophenylalanine site‑specifically into rHirudin at the 63 sites in an Escherichia coli system in response to the TAG codon. Following fusion with the gIII signal peptide and a hexahistidine tag, the modified protein was secreted into Luria‑Bertani culture medium and purified by nickel-nitrilotriacetic acid affinity chromatography following a gel filtration column. In a 200 ml flask, the yield of boronophenylalanine‑modified hirudin was 10 mg l‑1 and that of rHirudin was 19 mg l‑1. The authenticity of the purified proteins was verified using matrix-assisted laser desorption ionization time of flight mass spectroscopy and antithrombin activity assays. The results revealed that the antithrombin activity of the boronophenylalanine‑modified hirudin to human thrombin was more enhanced than that of rHirudin. The modified hirudin demonstrated stronger proliferation inhibiting ability on fibroblast L929 cells compared with that of rHirudin.

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Section: Expression Of Boronophenylalanine-modified Hirudin and Rhirudinsupporting

confidence: 74%

Improving the bioactivity of rHirudin with boronophenylalanine site-specific modification

Xin

Liu

Sun

2015

Molecular Medicine Reports

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“…In 1996, Rosenfeld et al synthetized rH in P. pastoris, and the expression level reached 1.5g/L (Rosenfeld et al, 1996). In recent decades, the hosts for expressing rH are also increasing, which vary from Bacillus subtilis (Chen et al, 2011) to Lactococcus lactis (Lv et al, 2012). rH can be produced not only by bacterial or yeast expression systems, but also by a cell-free protein synthesis approach, which can express rH with higher antithrombin activity (Wustenhagen et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

Pharmacological Activities and Mechanisms of Hirudin and Its Derivatives - A Review

et al. 2021

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Hirudin, an acidic polypeptide secreted by the salivary glands of Hirudo medicinalis (also known as “Shuizhi” in traditional Chinese medicine), is the strongest natural specific inhibitor of thrombin found so far. Hirudin has been demonstrated to possess potent anti-thrombotic effect in previous studies. Recently, increasing researches have focused on the anti-thrombotic activity of the derivatives of hirudin, mainly because these derivatives have stronger antithrombotic activity and lower bleeding risk. Additionally, various bioactivities of hirudin have been reported as well, including wound repair effect, anti-fibrosis effect, effect on diabetic complications, anti-tumor effect, anti-hyperuricemia effect, effect on cerebral hemorrhage, and others. Therefore, by collecting and summarizing publications from the recent two decades, the pharmacological activities, pharmacokinetics, novel preparations and derivatives, as well as toxicity of hirudin were systematically reviewed in this paper. In addition, the clinical application, the underlying mechanisms of pharmacological effects, the dose-effect relationship, and the development potential in new drug research of hirudin were discussed on the purpose of providing new ideas for application of hirudin in treating related diseases.

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Robust preparative-scale extracellular production of hirudin in Escherichia coli and its purification and characterization

Huang

Zhang

et al. 2012

Journal of Industrial Microbiology and Biotechnology

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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.

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Extracellular secretion of anticoagulant peptide hirudin in Lactococcus lactis using SP310mut2 signal peptide

Cited by 7 publications

References 15 publications

Improving the bioactivity of rHirudin with boronophenylalanine site-specific modification

Improving the bioactivity of rHirudin with boronophenylalanine site-specific modification

Pharmacological Activities and Mechanisms of Hirudin and Its Derivatives - A Review

Robust preparative-scale extracellular production of hirudin in Escherichia coli and its purification and characterization

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