Production of recombinant hirudin by high cell density fed-batch cultivations of a Saccharomyces cerevisiae strain: physiological considerations during the bioprocess design

Mendoza-Vega, O.; Hebert, C. Nancy; Brown, Stephen

doi:10.1016/0168-1656(94)90211-9

Cited by 23 publications

(4 citation statements)

References 32 publications

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“…Fiechter et al [4] devised a defined yeast medium for the aerobic growth of Schizosaccharomyces pombe (Dw medium) [4]. A modified version of the Dw medium has been employed successfully for S. cereuisiae fed-batch processes at high cell densities [102]. Our fed-batch results with the production process of recombinant hirudin have demonstrated a biomass production of 60 g 1-CDW and a high production level of recombinant protein of 50(1 mg 1-~ (Fig.…”

Section: Culture Medium Considerationsmentioning

confidence: 76%

“…In connection with this observation, we noticed a significant variation of the plasmid copy number in a 20-1 fed-batch culture of a hirudin-producing yeast strain (Table 3). An increase of the copy number of the 2->m based plasmid was accompanied by a loss of cell viability and a lower growth rate, whereas the recombinant protein yield and the plasmid containing cells (%P +) remained constant during the entire process [102]. Our experience with the cxpression of heterologous proteins using this 2-/xm based vector has not shown such a variation of plasmid copy number with a tightly regulated expression.…”

Section: Nature Of the Yeast Expression Cectorsmentioning

confidence: 79%

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Industrial production of heterologous proteins by fed-batch cultures of the yeast Saccharomyces cereuisiae

Mendoza-Vega

1994

FEMS Microbiology Reviews

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This review concerns the issues involved in the industrial development of fed-batch culture processes with Saccharomyces cerevisiae strains producing heterologous proteins. Most of process development considerations with fed-batch recombinant cultures are linked to the reliability and reproducibility of the process for manufacturing environments where quality assurance and quality control aspects are paramount. In this respect, the quality, safety and efficacy of complex biologically active molecules produced by recombinant techniques are strongly influenced by the genetic background of the host strain, genetic stability of the transformed strain and production process factors. An overview of the recent literature of these culture-related factors is coupled with our experience in yeast fed-batch process development for producing various therapeutic grade proteins. The discussion is based around three principal topics: genetics, microbial physiology and fed-batch process design. It includes the fundamental aspects of yeast strain physiology, the nature of the recombinant product, quality control aspects of the biological product, features of yeast expression vectors, expression and localization of recombinant products in transformed cells and fed-batch process considerations for the industrial production of Saccharomyces cerevisiae recombinant proteins. It is our purpose that this review will provide a comprehensive understanding of the fed-batch recombinant production processes and challenges commonly encountered during process development.

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Section: Culture Medium Considerationsmentioning

confidence: 76%

Section: Nature Of the Yeast Expression Cectorsmentioning

confidence: 79%

Industrial production of heterologous proteins by fed-batch cultures of the yeast Saccharomyces cereuisiae

Mendoza-Vega

1994

FEMS Microbiology Reviews

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“…That is comparable to the representative highyield of rHV2-Lys 47 in S. cerevisiae (500 mg l -1 ), which was reached by fed-batch cultivation for 57 h [14], and 1.5 g l -1 of secreted rHIR in methylotrophic yeast P. pastoris, which was achieved by fed-batch cultivation for 65 h after methanol induction [18,23]. However, it can be seen that the long cultivation period is absolutely needed for attaining high level of expression with the yeast system, and this will inevitably increase the preparation cost and complexity of manufacturing.…”

Section: Discussionmentioning

confidence: 65%

Enhanced secretion of adhesive recognition sequence containing hirudin III mutein in E. coli

Tan

Wu-tong

et al. 2007

Mol Biotechnol

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It has been previously shown that Escherichia coli L-asparaginase II (L-ASP) signal peptide is capable of being utilized to direct extracellular secretion of hirudin III (HV3) in shake flask. In this study HV3 muteins R33G34D35(S36)-HV3 were generated by introduction of adhesive recognition sequence RGD(S) into the non-functional region of HV3. The resultant recombinants were cultivated on 30 l bioreactor scale using L-ASP signal peptide expression system and the optimized fed-batch cultivation was well established. After cultivation for approximately 11 h the secreted product accumulated up to approximately 1 g l(-1), which means 17-fold increase in productivity compared to initial expression in shake flask. N-terminal analysis, pI measurement, and MALDI mass spectral analysis on mutein R33G34D35S36-HV3 confirmed the authenticity of the product. Compared to wild-type HV3 and R33G34D35HV3, the mutein R33G34D35S36-HV3 exhibits the improved pharmacological activity. Collectively, a novel secretion strategy using L-ASP signal peptide for the rapid, efficient and cost-effective production of HV3 mutein possessing improved pharmacological activity on bioreactor scale has been well established. Using this expression system downstream processing becomes very simple because secreted product is mature, soluble, active, and without N-terminal extension of Met, which is quite critical for most therapeutic protein to reduce the side effect in clinic use. Thus, it provides a promising alternative for extracellular production of other difficult-to-express protein for biopharmaceutical use.

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“…ºra> transformants were grown in liquid or solidified minimal medium YNBG [0.675% Yeast nitrogen base (Difco) 1% glucose]. For expression analysis liquid Ka¨ppeli medium (Mendoza-Vega et al 1994), with or without 2 mg/l thiamine, was used. Escherichia coli strain 5K (hsdR, mcrA) was used for cloning and was grown on LB medium supplemented with ampicillin (100 g/ml).…”

Section: Strains and Mediamentioning

confidence: 99%

Characterization of upstream activating sequences involved in activation and regulation of pho4 expression in Schizosaccharomyces pombe

Silvestre

Jacobs

1997

Mol Gen Genet

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In this study, a short region of the pho4 promoter located just upstream of the TATA box, called the upstream activating sequence (UAS), was shown to be responsible for both activation and regulation of pho4 expression in Schizosaccharomyces pombe. This cis-acting sequence is able to activate transcription when placed upstream of the minimal promoter of the constitutively expressed adh gene, and to ensure regulation by thiamine. The organisation of the pho4 promoter is remarkable in that UAS and TATA box are very closely associated. This proximity appears to be essential for efficient transcriptional regulation. Indeed, only slight variations in transcript levels were observed under derepression conditions when the UAS was moved away from the TATA box, while regulation of transcript levels appeared to be strongly affected. This observation suggests the existence of a negative regulatory element whose action requires very close association of UAS and TATA box. Surprisingly, when UAS was moved about 40 bp away from the adh TATA box, residual but very significant repression of gene expression by thiamine, which apparently does not result from transcriptional regulation, was revealed by measuring protein production. These data prompted us to hypothesize the existence of two distinct and cooperative mechanisms of regulation involving specific factor binding to the UAS, the first one controlling transcript levels and the second acting posttranscriptionally.

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Production of recombinant hirudin by high cell density fed-batch cultivations of a Saccharomyces cerevisiae strain: physiological considerations during the bioprocess design

Cited by 23 publications

References 32 publications

Industrial production of heterologous proteins by fed-batch cultures of the yeast Saccharomyces cereuisiae

Industrial production of heterologous proteins by fed-batch cultures of the yeast Saccharomyces cereuisiae

Enhanced secretion of adhesive recognition sequence containing hirudin III mutein in E. coli

Characterization of upstream activating sequences involved in activation and regulation of pho4 expression in Schizosaccharomyces pombe

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