Influence of specific growth rate over the secretory expression of recombinant potato carboxypeptidase inhibitor in fed-batch cultures of Escherichia coli

Puertas, Juan Miguel; Ruiz, Jordi; Vega, Mónica Rodríguez de la; Lorenzo, Júlia; Caminal, Glòria; González, Gerardo

doi:10.1016/j.procbio.2010.04.024

Cited by 15 publications

(13 citation statements)

References 38 publications

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“…It has been reported that specific growth rate (µ) affects the amount of soluble excreted target product and that secretion of target product to the periplasmic space and culture media is increased at µ values lower than 0.25 h -1 before induction, in fed batch fermentation process with an exponential feeding strategy (Puertas et al 2010). In the present work, the specific growth rate at the moment of induction was 0.19 h -1…”

Section: Resultsmentioning

confidence: 52%

Production of antibody fragment (Fab) throughout Escherichia coli fed-batch fermentation process: Changes in titre, location and form of product

Jalalirad¹

2013

Electron. J. Biotechnol.

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Background: Recombinant proteins, including antibodies and antibody fragments, often contain disulfide bond bridges that are necessary for their folding, stability and function. Production of disulfidebond-containing proteins in the periplasm of Escherichia coli has been very useful, due to unique characteristics of the periplasm, for obtaining fully active and correctly folded products and for alleviating downstream processing. Results: In this study, fed-batch cultivation of Escherichia coli (E. coli) for production of Fab D1.3, which is an anti-hen egg white lysozyme (HEWL) antibody fragment was carried out at 37ºC, and the bacterial cells were induced by adding 0.1 mM IPTG to the culture medium. Fermentor was sampled over the course of fermentation; the bacterial cells were centrifugally separated from the culture broth and subjected to osmotic shock (with excluding HEWL) and sonication procedures. The resulting fractions were analysed for Fab using a combination of ELISA, SDS-PAGE and Western blotting and changes in product titre, location, and form was assessed throughout growth. It was shown that osmotic shock released the Fab from the periplasm very efficiently and its efficacy was 20-45% more than sonication. This study demonstrates that, at high cell density cultivation in fermentor, target product can appear inside and outside the cells, depending on the time of induction. The maximum amount of Fab (47 mg/l) in the periplasm was reached at 14 hrs cultivation (4 hrs post induction), being suitable time for cell harvest, selective periplasmic extraction and downstream capture. The Fab increasingly leaked into the culture medium, and reached its maximum culture medium titre of ~78 mg/l after 6 hrs post induction. After 16 hrs cultivation (6 hrs post induction) the amount of Fab remained constant in different locations within and outside the cells. Western blot analysis of cell fractions showed that certain amount of the Fab was also produced in the cells as insoluble form. Conclusions: In this work we showed that the production of Fab in the periplasm during high cell density cultivation of E. coli in fermentor can be challenging as the product may appear in various locations within and outside the cells. To exploit the advantages of the periplasmic expression systems for purification in downstream processing, bacterial cells should be harvested when they maintain.

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Section: Resultsmentioning

confidence: 52%

Production of antibody fragment (Fab) throughout Escherichia coli fed-batch fermentation process: Changes in titre, location and form of product

Jalalirad¹

2013

Electron. J. Biotechnol.

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“…The ability to maintain cells in a particular metabolic or physiological state related to μ, promoting a particular metabolism for the production of secondary metabolites, such as prodigiosins (Chang et al 2011), is of particular relevance to biotechnology and pharmaceutical industries. Generally speaking, the specific productivity, recombinant protein production rate, and secretory expression in carbon-limited fed-batch cultures is often inversely correlated to the specific growth rate of the microorganism (Jenzsch et al 2006)-hence the importance of maintaining low specific growth rates, at least during protein expression phase, to maximize product yield (Panda et al 1999;Hellwig et al 2001;Priego-Jimenez et al 2005;Ren and Yuan 2005;Jenzsch et al 2006;Babaeipour et al 2007;Min et al 2010;Puertas et al 2010).…”

Section: Product Formation Rate and Product Quantitymentioning

confidence: 99%

“…RNA synthesis and content within cells is reported to increase with the specific growth rate (Lange and Heijnen 2001;Cox 2004). Plasmid stability, of particular interest in the recombinant protein production using E. coli or Bacillus subtilis, has also been associated with the specific growth rate (Babaeipour et al 2007;Demain and Vaishnav 2009;Puertas et al 2010). The protein synthesizing ability of cells, also depending on μ, is of particular importance for recombinant protein production.…”

Section: Product Formation Rate and Product Quantitymentioning

confidence: 99%

“…The quality of the product is, at least for the production of therapeutics, even more important than the titer. A number of studies have highlighted the effect of changes in the specific growth rate on the glycosylation pattern (Jungo et al 2007) or on the secretory expression (Puertas et al 2010) of certain recombinant proteins. Characterization of product quality is predominately carried out off-line, frequently once the process is finished.…”

Section: Product Qualitymentioning

confidence: 99%

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Real-time monitoring and control of microbial bioprocesses with focus on the specific growth rate: current state and perspectives

Schuler

Marison

2012

Appl Microbiol Biotechnol

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Understanding the growth characteristics of microorganisms is an essential step in bioprocessing, not only because product formation may be growth-associated but also because they might influence cell physiology and thereby product quality. The specific growth rate, a key variable of many bioprocesses, cannot be measured directly and relies on the estimation through other measurable variables such as biomass, substrate, or product concentrations. Techniques for real-time estimation of the specific growth rate in microbial fed-batch cultures are discussed in the present paper. The advantages and limitations of different models and various monitoring techniques are discussed, highlighting the importance of the specific growth rate in the development of fast, reliable, and robust processes for the production of high-value products such as recombinant proteins.

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“…Once the glucose was depleted, addition of feedstock containing glycerol was started according to an exponential feed profile, which allowed fixing the specific growth rate at l = 0.1 h -1 by limiting the carbon source. This cultivation protocol and media used are thoroughly described elsewhere [18].…”

Section: Bioreactor Cultivation Conditionsmentioning

confidence: 99%

Expression of metallocarboxypeptidase inhibitors in Escherichia coli: effect of cysteine content and protein size in the secretory production of disulfide-bridged proteins

Puertas

Caminal

González

2011

J Ind Microbiol Biotechnol

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Metallocarboxypeptidase inhibitors are proteins with possible applications in biomedicine given their properties as anticoagulant and antitumoral factors. They are small, eukaryotic polypeptides comprising several disulfide bridges, which makes them hard to express in inexpensive bacterial hosts. In this work, three of them were produced in high-cell-density cultures of Escherichia coli: PCI (39 residues and three bridges), LCI (66 residues and four bridges) and TCI (75 residues and six bridges). The genes coding for the mentioned inhibitors were cloned in an arabinose-inducible plasmid fused to the signal peptide of DsbA in order to have them secreted and grant the formation of the bridges. The trigger-factor defective strain KTD101 was used as the expression host. The resulting recombinant strains were cultured in fed-batch mode employing minimal media and an exponential feed profile, keeping the specific growth rate at μ = 0.1 h(-1) by limitation of the fed carbon source (glycerol). Between 380 and 540 mg l(-1) of active inhibitors were obtained in both the periplasmic extracts and extracellular media of the cultures. Later on, excretion was enhanced using a cell permeabilization treatment, allowing the recovery of over 80% of the products from the extracellular fraction. Protein yields were found to be inversely proportional to cysteine content of the inhibitor, whereas protein excretion rates were inversely proportional to the protein size. Overall, these results offer insight into the secretory production of active disulfide-bridged proteins in high-cell-density cultures of E. coli.

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Influence of specific growth rate over the secretory expression of recombinant potato carboxypeptidase inhibitor in fed-batch cultures of Escherichia coli

Cited by 15 publications

References 38 publications

Production of antibody fragment (Fab) throughout Escherichia coli fed-batch fermentation process: Changes in titre, location and form of product

Production of antibody fragment (Fab) throughout Escherichia coli fed-batch fermentation process: Changes in titre, location and form of product

Real-time monitoring and control of microbial bioprocesses with focus on the specific growth rate: current state and perspectives

Expression of metallocarboxypeptidase inhibitors in Escherichia coli: effect of cysteine content and protein size in the secretory production of disulfide-bridged proteins

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