Process development for the production of recombinant hirudin in Saccharomyces cerevisiae: from upstream to downstream

Sohn, Jae Hak; Choi, Eui Sung; Chung, Bong Hyun; Youn, Duk Joong; Seo, Jin Ho; Rhee, Shin Hyung

doi:10.1016/0032-9592(94)00062-x

Cited by 23 publications

(8 citation statements)

References 9 publications

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“…The plasmids were maintained and propagated in E. coli TOP10 in accordance with the methods described by Sambrook et al [34]. The pMYO50 plasmid harboring the E. coli heat-labile enterotoxin B subunit gene (sLTB) was utilized [18], and S. cerevisiae 2805 (MATα pep4::HIS3 prb1-δ Can1 GAL2 his3 ura3-52) was used as the recipient cell for LTB production [39].…”

Section: Strains and Culture Conditionsmentioning

confidence: 99%

Expression of Functional Pentameric Heat-Labile Enterotoxin B Subunit of Escherichia coli in Saccharomyces cerevisiae

Lim

Kim²,

Chung³

et al. 2009

J.Microbiol.Biotechnol

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Although the Escherichia coli heat-labile enterotoxin B subunit (LTB) has already been expressed in several different systems, including prokaryotic and eukaryotic organisms, studies regarding the synthesis of LTB into oligomeric structures of pentameric size in the budding yeast Saccharomyces cerevisiae have been limited. Therefore, this study used a functional signal peptide of the amylase 1A protein from rice to direct the yeast-expressed LTB towards the endoplasmic reticulum to oligomerize with the expected pentameric size. The expression and assembly of the recombinant LTB were confirmed in both the cellfree extract and culture media of the recombinant strain using a Western blot analysis. The binding of the LTB pentamers to intestinal epithelial cell membrane glycolipid receptors was further verified using a GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA). On the basis of the GM1-ELISA results, pentameric LTB proteins comprised approximately 0.5-2.0% of the total soluble proteins, and the maximum quantity of secreted LTB was estimated to be 3 mg/l after a 3-day cultivation period. Consequently, the synthesis of LTB monomers and their assembly into biologically active oligomers in a recombinant S. cerevisiae strain demonstrated the feasibility of using a GRAS microorganism-based adjuvant, as well as the development of carriers against mucosal disease.

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Section: Strains and Culture Conditionsmentioning

confidence: 99%

Expression of Functional Pentameric Heat-Labile Enterotoxin B Subunit of Escherichia coli in Saccharomyces cerevisiae

Lim

Kim²,

Chung³

et al. 2009

J.Microbiol.Biotechnol

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“…cerevisiae to get the efficient production of rHV2 with the expression level of 21 ATU/ml (Loison et al, 1988). Furthermore, Sohn et al optimized the expression vector and fermentation conditions, enabling the expression level of recombinant hirudin to reach to 460 mg/L (Sohn et al, 1995). In 1996, Rosenfeld et al synthetized rH in P. pastoris, and the expression level reached 1.5g/L (Rosenfeld et al, 1996).…”

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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“…Yeast systems have proven more suitable for production of rhir on an industrial scale. Expression in S. cerevisiae of hirudin fused to the α-mat-ing factor secretory prepro sequence has yielded levels as high as 40-460 mg/l culture and allowed easy purification of rhir from the culture filtrate [Riehl-Bellon et al, 1989;Sohn et al, 1995]. The specific activity obtained in these studies was reported to be near the value of desulphated hirudin.…”

Section: Hirudin As a Model Pharmaceutical Proteinmentioning

confidence: 92%

Molecular farming in plants: Oilseeds as vehicles for the production of pharmaceutical proteins

1997

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The advantages and limitations of plant‐based systems for the production of recombinant biopharmaceutical proteins are discussed in the context of other host production systems presently available. A novel approach for the purification of recombinant proteins expressed in oilseeds is described that is based on the targeting of these proteins to seed oil bodies. The utility of this approach is demonstrated by the purification of the anticoagulant protein hirudin, produced in seeds of Brassica napus. Drug Dev. Res. 42:172–181, 1997. © 1997 Wiley‐Liss, Inc.

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Process development for the production of recombinant hirudin in Saccharomyces cerevisiae: from upstream to downstream

Cited by 23 publications

References 9 publications

Expression of Functional Pentameric Heat-Labile Enterotoxin B Subunit of Escherichia coli in Saccharomyces cerevisiae

Expression of Functional Pentameric Heat-Labile Enterotoxin B Subunit of Escherichia coli in Saccharomyces cerevisiae

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

Molecular farming in plants: Oilseeds as vehicles for the production of pharmaceutical proteins

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