Generation of a histidine-tagged antibotulinum toxin antibody fragment in E. coli : effects of post-induction temperature on yield and IMAC binding-affinity

Bentley, William E.; Madurawe, Rapti D.; Gill, Ryan T.; Shiloach, M; Chase, Terry E.; Pulliam-Holoman, T R; Valdés, James J.

doi:10.1038/sj.jim.2900577

Cited by 5 publications

(4 citation statements)

References 12 publications

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“…Further, all plasmids contained the ampicillin resistance gene ( ;-lactamase). These proteins were chloramphenicol acetyl-transferase (CAT; Bentley et al, 1990), human interleukin-2 (IL-2; Pham et al, 1999), an antibotulinum toxin antibody fragment (btFab; Bentley et al, 1999), the coat protein of the tobacco mosaic virus (TMVCP; Dardick and Culver, 1997), and viral protein 5 (VP5) from segment A of double-stranded RNA infectious bursal disease virus (IBDV) (Mundt et al, 1995).…”

Section: Introductionmentioning

confidence: 99%

A Comparative Study of Global Stress Gene Regulation in Response to Overexpression of Recombinant Proteins in Escherichia coli

Gill

Valdés

Bentley

2000

Metabolic Engineering

117

111

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Section: Introductionmentioning

confidence: 99%

A Comparative Study of Global Stress Gene Regulation in Response to Overexpression of Recombinant Proteins in Escherichia coli

Gill

Valdés

Bentley

2000

Metabolic Engineering

117

111

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“…It is well‐known that the optimum temperature for E. coli culture is 37 °C. However, Bentley et al (10) studied the effects of post‐induction temperature for fermentation culture on maximal bt‐Fab antibody production. They tested three different post‐induction temperatures (i.e., 20, 30, and 37 °C) and found that the profiles of bt‐Fab antibody during the post‐induction period for both 25 and 30 °C were detected, while bt‐Fab was not detected at 37 °C.…”

Section: Resultsmentioning

confidence: 99%

“…The addition of a histidine tail facilitated purification by immobilized metal affinity chromatography (IMAC). Expression and purification of the recombinant bt‐Fab antibody from microbial fermentation is less expensive, easier, and less time‐consuming than production of Mabs by hybridoma cell culture (8 − 10). Given that the main aim of this study was to maximize bt‐Fab antibody production, optimization of the fermentation process could be conducted either by changing one factor at a time or by varying several factors at the same time and looking for interactions using statistical analysis.…”

Section: Introductionmentioning

confidence: 99%

Rapid Optimization of Antibotulinum Toxin Antibody Fragment Production by an Integral Approach Utilizing RC-SELDI Mass Spectrometry and Statistical Design

Park

Bradbury²,

Kragl

et al. 2006

Biotechnol. Prog.

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A process for the rapid development and optimization of the fermentation process for an antibotulinum neurotoxin antibody fragment (bt-Fab) production expressed in Escherichia coli was achieved via a high-throughput process proteomics and statistical experimental design. This process, using retentate chromatography-surface enhanced laser desorption/ionization mass spectrometry (RC-SELDI MS), was employed for identifying and quantifying bt-Fab antibody in complex biological samples for the optimization of microbial fermentation conditions. Five variables (type of culture media, glycerol concentration, post-induction temperature, IPTG concentration, and incubation time after induction) were statistically combined using an experimental 2(5)(-1) fractional factorial design and tested for their effects on maximal bt-Fab antibody production. When the effects of individual variables and their interactions were assessed, type of media and post-induction temperature showed statistically significant increase in yield of the fermentation process for the maximal bt-Fab antibody production. This study establishes an integral approach as a valuable tool for the rapid development of manufacturing processes for producing various biological materials. To verify the RC-SELDI MS method, a Fab-specific immuno-affinity HPLC assay developed here was also employed for the quantification of the bt-Fab antibody in crude lysate samples obtained during the fermentation optimization process. Similar results were obtained.

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“…Also, it was assumed that α and k d are functions of inducer concentration: where I is the IPTG concentration (mM) and k α m , K α S , K α I , k dm , K dS , K dI are constants. The form of these expressions enables a maximum expression rate as function of inducer, which is commonly observed as a result of the metabolic burden associated with protein expression (15−17). When cobalt ion is added, the activity increases.…”

Section: Methodsmentioning

confidence: 99%

Effects of in Situ Cobalt Ion Addition on the Activity of a GFP-OPH Fusion Protein: The Fermentation Kinetics

Valdés

Bentley

2001

Biotechnol. Prog.

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The effects of cobalt ion addition and inducer concentration were studied in the fermentation of E. coli BL21 expressing a GFP (green fluorescent protein)-OPH (organophosphorus hydrolase) fusion protein. It was found that cobalt ion addition improved the OPH activity significantly. When 2 mM of CoCl(2) was supplied during the IPTG-induction phase, OPH activity was enhanced approximately 10-fold compared to the case without cobalt or by the addition of cobalt to the cell extracts. Results indicate, therefore, that incorporation of the cobalt during synthesis is needed for enhanced activity. Also, the maximum OPH activity was not linearly related to inducer concentration. A mathematical model was then constructed to simulate these phenomena. Model parameters were determined by constrained least-squares and optimal IPTG and cobalt addition concentrations were obtained, pinpointing the conditions for the maximum productivity. Finally, the GFP fluorescence intensity was found linear to the OPH activity in each fermentation, demonstrating the function of GFP for monitoring its fusion partner's quantity in the bioreactor.

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Generation of a histidine-tagged antibotulinum toxin antibody fragment in E. coli : effects of post-induction temperature on yield and IMAC binding-affinity

Cited by 5 publications

References 12 publications

A Comparative Study of Global Stress Gene Regulation in Response to Overexpression of Recombinant Proteins in Escherichia coli

A Comparative Study of Global Stress Gene Regulation in Response to Overexpression of Recombinant Proteins in Escherichia coli

Rapid Optimization of Antibotulinum Toxin Antibody Fragment Production by an Integral Approach Utilizing RC-SELDI Mass Spectrometry and Statistical Design

Effects of in Situ Cobalt Ion Addition on the Activity of a GFP-OPH Fusion Protein: The Fermentation Kinetics

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