Evaluation of a pH-stat feeding strategy on the production and recovery of Fab’ fragments from E. coli

García-Arrazola, Roeb; Siu, Sun Chau; Chan, G.H.T.; Buchanan, Ian; Doyle, Billy; Titchener‐Hooker, Nigel J.; Baganz, Frank

doi:10.1016/j.bej.2005.01.003

Cited by 25 publications

(18 citation statements)

References 26 publications

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“…Cultures were grown in LB broth (10 gÁL À1 bacto-tryptone, 5 gÁL À1 yeast extract, 10 g L À1 sodium chloride) in the presence of ampicillin (100 lgÁmL À1 ) and kanamycin (50 lgÁmL À1 ), where applicable. The defined medium was used for fermentation experiments [31], and was supplemented with ampicillin (100 lgÁmL À1 ) and kanamycin (50 lgÁmL À1 ) where applicable.…”

Section: Methodsmentioning

confidence: 99%

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High‐level secretion of a recombinant protein to the culture medium with a Bacillus subtilis twin‐arginine translocation system in Escherichia coli

et al. 2013

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The twin‐arginine translocation (Tat) system transports folded proteins across the plasma membrane in bacteria, and heterologous proteins can be exported by this pathway if a Tat‐type signal peptide is present at the N‐terminus. The system thus has potential for biopharmaceutical production in Escherichia coli, where export to the periplasm is often a favoured approach. Previous studies have shown that E. coli cells can export high levels of protein by the Tat pathway, and the protein product accummulates almost exclusively in the periplasm. In this study, we analysed E. coli cells that express the Bacillus subtilis TatAdCd system in place of the native TatABC system. We show that a heterologous model protein, comprising the TorA signal peptide linked to green fluorescent protein (TorA–GFP), is efficiently exported by the TatAdCd system. However, whereas the GFP is exported initially to the periplasm during batch fermentation, the mature protein is increasingly found in the extracellular culture medium. By the end of a 16‐h fermentation, ~ 90% of exported GFP is present in the medium as active mature protein. The total protein profiles of the medium and periplasm are essentially identical, confirming that the outer membrane becomes leaky during the fermentation process. The cells are otherwise intact, and there is no large‐scale release of cytoplasmic contents. Export levels are relatively high, with ~ 0.35 g GFP·L−1 culture present in the medium. This system thus offers a means of producing recombinant protein in E. coli and harvesting directly from the medium, with potential advantages in terms of ease of purification and downstream processing.

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Section: Methodsmentioning

confidence: 99%

“…The defined medium was used for fermentation experiments [31], and was supplemented with ampicillin (100 lgÁmL…”

mentioning

confidence: 99%

High‐level secretion of a recombinant protein to the culture medium with a Bacillus subtilis twin‐arginine translocation system in Escherichia coli

et al. 2013

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“…To prevent foaming, 100 lL bolus additions of a 10% (v/v) PPG 2000 solution were performed at regular intervals from inoculation to induction using the liquid handler. The microbioreactors were supplemented with 15 mM magnesium sulfate (2.0 M stock) and 35 mM sodium phosphate (2.0 M stock, pH 6.5 at 258C) when the cells grew to an OD 600 nm of 30-40. Feeding and induction were triggered on an individual vessel basis by way of a control loop observing an increase in pH when the main carbon source was exhausted [26][27][28][29] ; cells were fed with 40% (w/w) glycerol at 3.2 mL L 21 h 21 using the pumped liquid delivery system and induced using the liquid handler in a single bolus addition to a final concentration in the vessels of 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG). Cell growth was monitored off-line by optical density measurements at 600 nm.…”

Section: Fed-batch Fermentationsmentioning

confidence: 99%

High throughput automated microbial bioreactor system used for clone selection and rapid scale‐down process optimization

Velez-Suberbie

Betts

Walker

et al. 2017

Biotechnology Progress

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High throughput automated fermentation systems have become a useful tool in early bioprocess development. In this study, we investigated a 24 x 15 mL single use microbioreactor system, ambr 15f, designed for microbial culture. We compared the fed‐batch growth and production capabilities of this system for two Escherichia coli strains, BL21 (DE3) and MC4100, and two industrially relevant molecules, hGH and scFv. In addition, different carbon sources were tested using bolus, linear or exponential feeding strategies, showing the capacity of the ambr 15f system to handle automated feeding. We used power per unit volume (P/V) as a scale criterion to compare the ambr 15f with 1 L stirred bioreactors which were previously scaled‐up to 20 L with a different biological system, thus showing a potential 1,300 fold scale comparability in terms of both growth and product yield. By exposing the cells grown in the ambr 15f system to a level of shear expected in an industrial centrifuge, we determined that the cells are as robust as those from a bench scale bioreactor. These results provide evidence that the ambr 15f system is an efficient high throughput microbial system that can be used for strain and molecule selection as well as rapid scale‐up. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:58–68, 2018

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“…[6,7] High cell density cultivation (HCDC) has been developed to increase production of recombinant proteins in appropriate amounts for research, and clinical and industrial approaches. [5,8] Fed-batch cultivation is an effective method that is used widely to achieve high cell density. [9] The addition of concentrated medium in this process can prevent nutrient limitation and prolong the growth phase, leading to higher biomass and product concentrations [10] .…”

Section: Introductionmentioning

confidence: 99%

“…This implied that appropriate concentrations of nitrogen and minerals were moved at vertex 11-16, where the response of dry cell weight was excessive(Figure 2). The lowest responses (vertices[5][6][7][8][9][10][11] represented the upper levels of both nutrients. In contrast, the concentration of glycerol was increased while the tryptone concentration was reduced at the optimum point.…”

mentioning

confidence: 99%

Multiparameter optimization method and enhanced production of secreted recombinant single-chain variable fragment against the HIV-1 P17 protein fromEscherichia coliby fed-batch fermentation

Paopang

Kasinrerk

Tayapiwatana

et al. 2015

Preparative Biochemistry and Biotechnology

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The single-chain fragment variable (scFv) was used to produce a completely functional antigen-binding fragment in bacterial systems. The advancements in antibody engineering have simplified the method of producing Fv fragments and made it more efficient and generally relevant. In a previous study, the scFv anti HIV-1 P17 protein was produced by a batch production system, optimized by the sequential simplex optimization method. This study continued that work in order to enhance secreted scFv production by fed-batch cultivation, which supported high volumetric productivity and provided a large amount of scFvs for diagnostic and therapeutic research. The developments in cell culture media and process parameter settings were required to realize the maximum production of cells. This study investigated the combined optimization methods, Plackett-Burman design (PBD) and sequential simplex optimization, with the aim of optimize feed medium. Fed-batch cultivation with an optimal feeding rate was determined. The result demonstrated that a 20-mL/hr feeding rate of the optimized medium can increase cell growth, total protein production, and scFv anti-p17 activity by 4.43, 1.48, and 6.5 times more than batch cultivation, respectively. The combined optimization method demonstrated novel power tools for the optimization strategy of multiparameter experiments.

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Evaluation of a pH-stat feeding strategy on the production and recovery of Fab’ fragments from E. coli

Cited by 25 publications

References 26 publications

High‐level secretion of a recombinant protein to the culture medium with a Bacillus subtilis twin‐arginine translocation system in Escherichia coli

High‐level secretion of a recombinant protein to the culture medium with a Bacillus subtilis twin‐arginine translocation system in Escherichia coli

High throughput automated microbial bioreactor system used for clone selection and rapid scale‐down process optimization

Multiparameter optimization method and enhanced production of secreted recombinant single-chain variable fragment against the HIV-1 P17 protein fromEscherichia coliby fed-batch fermentation

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