Production of recombinant proteins by high cell density culture of Escherichia coli

Choi, Jaehyouk; Keum, Ki Chang; Lee, Sang Yup

doi:10.1016/j.ces.2005.03.031

Cited by 281 publications

(205 citation statements)

References 63 publications

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“…The development of recombinant DNA technology allows enzyme production in large quantities with similar properties to the native form (Cohen et al 1973;Choi et al 2006). A previous study successfully isolated and characterized the bcfd1 gene that encoded dehalogenase enzyme from Bacillus cereus IndB1 obtained from the Indonesian Agriculture Research and Development Association (Arif 2013).…”

Section: Introduc Onmentioning

confidence: 99%

Cloning and expression of haloacid dehalogenase gene from Bacillus cereus IndB1

Ratnaningsih

Idris²

2018

Indones. J. Biotechnol.

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Organohalogen compounds, widely used as pes cides in agriculture and solvents in the industrial sector, cause environmental pollu on and health problems due to their toxicity and persistence. Numerous studies have been conducted on the biodegrada on of organohalogen compounds, with many focusing on the use of dehalogenase from bacteria. Haloacid dehalogenase is a group of enzymes that cleaves the carbon-halogen bond in halogenated alipha c acids. In a previous study, the bcfd1 gene encoded haloacid dehalogenase from Bacillus cereus IndB1 was successfully isolated and characterized. This research aimed to create an expression system of the bcfd1 gene by subcloning this gene into pET expression vector and to overexpress the gene in Escherichia coli BL21 (DE3). In addi on, the recombinant protein was characterized to gain a be er understanding of the cataly c ac on of this enzyme. A high expression of bcfd1 was obtained by inducing the culture at OD 550 0.8-1.0 using 0.01 mM IPTG as determined by SDS-PAGE. Zymogram analysis proved that the recombinant protein possessed dehalogenase ac vity. Bcfd1 ac vity toward monochloroace c acid (MCA) showed specific ac vity of 37 U/mg at 30°C, pH 9. The predicted ter ary structure of Bcfd1 was es mated has conserved α/ß hydrolase folding mo f for haloacid dehalogenase superfamily.

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Section: Introduc Onmentioning

confidence: 99%

Cloning and expression of haloacid dehalogenase gene from Bacillus cereus IndB1

Ratnaningsih

Idris²

2018

Indones. J. Biotechnol.

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“…Plasmid loss occurs significantly over the post-exponential growth phase during protein expression period ( figure 3(a)). Decrease in the plasmid stability after induction in both scales was owing to increased metabolic pressure resulting from over-expression of recombinant protein and therefore decrease of specific growth rate (Lee, 2006: Babaeipour et al, 2007 thus antibiotic was used while induction of the process.…”

Section: Discussionmentioning

confidence: 99%

“…Volumetric productivity enhancement of recombinant microorganisms fermentation process usually is achieved in high cell density and more specific productivity (amount of production per cell) that can be obtained through fed-batch cultivation (Matsui et al, 2006;Chung et al, 2006). Hence, to achieve a successful high cell density culture (HCDC), selection of nutrient feeding strategy is critical issue (Tripathi, 2009;Choi, 2006). The exponential feeding strategy lets cells grow at a specific growth rate (Tripathi, 2009;Choi, 2006;Thiry and Cingolani., 2002) and adjust glucose concentration near zero without fluctuation thus causes minimum perturbation on cellular carbon metabolism (Choi, 2006).…”

Section: Introductionmentioning

confidence: 99%

“…Hence, to achieve a successful high cell density culture (HCDC), selection of nutrient feeding strategy is critical issue (Tripathi, 2009;Choi, 2006). The exponential feeding strategy lets cells grow at a specific growth rate (Tripathi, 2009;Choi, 2006;Thiry and Cingolani., 2002) and adjust glucose concentration near zero without fluctuation thus causes minimum perturbation on cellular carbon metabolism (Choi, 2006). HCDC has several drawbacks too, including limited availability of dissolved oxygen, acetate formation and reduced mixing efficiency which amplified by scale-up (Choi, 2006;Thiry et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

“…The exponential feeding strategy lets cells grow at a specific growth rate (Tripathi, 2009;Choi, 2006;Thiry and Cingolani., 2002) and adjust glucose concentration near zero without fluctuation thus causes minimum perturbation on cellular carbon metabolism (Choi, 2006). HCDC has several drawbacks too, including limited availability of dissolved oxygen, acetate formation and reduced mixing efficiency which amplified by scale-up (Choi, 2006;Thiry et al, 2002). Developing of proper HCDC conditions requires in the fermentation scale up, because of heat and mass transfer problems which arise in the large scale bioreactors (Thiry et al, 2002;Hewitt et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Bench-scale Overproduction and Purification of recombinant GCSF in Escherichia coli fed-batch process

2017

J App Pharm Sci

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Fermentation process scale up can change the hydrodynamic conditions of recombinant E. coli cultivation bioreactor and reduce productivity in the same operating conditions that have been developed at lab scale. In this study, an efficient strategy was used to scale up recombinant human GCSF production in the fed -batch culture of E. coli. Heterologous production of human GCSF was done in the fed-batch culture of Escherichia coli BL21 (DE3) with feeding strategy of maximum attainable specific growth rate. Then, fed -batch process was scaled up based on geometrical analogy and constant DO. Rh-GCSF was overexpressed as inclusion body (IB). Then IBs were released by cell disruption afterward solubilized and refolded properly. Eventually, cation exchanger chromatography was applied for the rh-GCSF purification. By induction at cell density of 75 g dry cell weight per l (g dcw/l), final cell density and rh-GCSF concentration in both scales were 124 and 114 g dcw/l, and 22 and 19 g protein/l obtained, respectively. Recovery yield of purification process was 400 mg purified gcsf per 1 g IB and recombinant protein purity was greater than 99%. Insignificant decrease of the biomass and rh-GCSF production (less than 10 percent) during scale up indicates efficiency of scale up method.

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Expression Hosts for Enzyme Discovery and Production

Andryushkova

Glieder

2009

Biocatalysis for the Pharmaceutical Industry

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Production of recombinant proteins by high cell density culture of Escherichia coli

Cited by 281 publications

References 63 publications

Cloning and expression of haloacid dehalogenase gene from Bacillus cereus IndB1

Cloning and expression of haloacid dehalogenase gene from Bacillus cereus IndB1

Bench-scale Overproduction and Purification of recombinant GCSF in Escherichia coli fed-batch process

Expression Hosts for Enzyme Discovery and Production

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