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Fontani, Silvia; Niccolai, Andrea; Kapat, Arnab; Olivieri, R.

doi:10.1023/a:1025104119260

Cited by 9 publications

(2 citation statements)

References 9 publications

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“…Densitometry is the technique normally employed to determine protein concentration [9,11,22,27,28,32,34,36,40,45,51,53] when it is not possible to evaluate it by means of enzyme activity assays.…”

Section: Resultsmentioning

confidence: 99%

Influence of induction conditions on the expression of carbazole dioxygenase components (CarAa, CarAc, and CarAd) from Pseudomonas stutzeri in recombinant Escherichia coli using experimental design

Larentis

Sampaio

Martins

et al. 2010

J Ind Microbiol Biotechnol

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Carbazole 1,9a-dioxygenase (CarA), the first enzyme in the carbazole degradation pathway used by Pseudomonas sp., was expressed in E. coli under different conditions defined by experimental design. This enzyme depends on the coexistence of three components containing [2Fe-2S] clusters: CarAa, CarAc, and CarAd. The catalytic site is present in CarAa. The genes corresponding to components of carbazole 1,9a-dioxygenase from P. stutzeri were cloned and expressed by salt induction in E. coli BL21-SI (a host that allows the enhancement of overexpressed proteins in the soluble fraction), using the vector pDEST™14. The expression of these proteins was performed under different induction conditions (cell concentration, temperature, and time), with the help of two-level factorial design. Cell concentration at induction (measured by absorbance at 600 nm) was tested at 0.5 and 0.8. After salt induction, expression was performed at 30 and 37°C, for 4 h and 24 h. Protein expression was evaluated by densitometry analysis. Expression of CarAa was enhanced by induction at a lower cell concentration and temperature and over a longer time, according to the analysis of the experimental design results. The results were validated at Abs (ind) = 0.3, 25°C, and 24 h, at which CarAa expression was three times higher than under the standard condition. The behavior of CarAc and CarAd was the inverse, with the best co-expression condition tested being the standard one (Abs (ind) = 0.5, T = 37°C, and t = 4 h). The functionality of the proteins expressed in E. coli was confirmed by the degradation of 20 ppm carbazole.

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Section: Resultsmentioning

confidence: 99%

Influence of induction conditions on the expression of carbazole dioxygenase components (CarAa, CarAc, and CarAd) from Pseudomonas stutzeri in recombinant Escherichia coli using experimental design

Larentis

Sampaio

Martins

et al. 2010

J Ind Microbiol Biotechnol

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“…However, the prokaryotic systems remains the most attractive hosts due to their low cost, high productivity and rapid production rates [30]. Prokaryotic heterologous protein expression is mainly carried out in the bacteria E. coli , although increasingly the Bacillus species are being employed [41,42,43]. Drawbacks of prokaryotic expression systems include poor protein quality, due to the inability of prokaryotic cells to carry out post-translational modifications such as glycosylation, the presence of toxic cell wall pyrogens, along with the formation of inclusion bodies resulting in aggregated and insoluble heterologous protein [44].…”

Section: Production Of Recombinant Proteins In a Prokaryotic Exprementioning

confidence: 99%

The Goldilocks Approach: A Review of Employing Design of Experiments in Prokaryotic Recombinant Protein Production

Uhoraningoga¹,

Kinsella²,

Henehan³

et al. 2018

Bioengineering

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The production of high yields of soluble recombinant protein is one of the main objectives of protein biotechnology. Several factors, such as expression system, vector, host, media composition and induction conditions can influence recombinant protein yield. Identifying the most important factors for optimum protein expression may involve significant investment of time and considerable cost. To address this problem, statistical models such as Design of Experiments (DoE) have been used to optimise recombinant protein production. This review examines the application of DoE in the production of recombinant proteins in prokaryotic expression systems with specific emphasis on media composition and culture conditions. The review examines the most commonly used DoE screening and optimisation designs. It provides examples of DoE applied to optimisation of media and culture conditions.

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The Taguchi methodology as a statistical tool for biotechnological applications: A critical appraisal

Rs¹,

Kumar

et al. 2008

Biotechnology Journal

392

220

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Success in experiments and/or technology mainly depends on a properly designed process or product. The traditional method of process optimization involves the study of one variable at a time, which requires a number of combinations of experiments that are time, cost and labor intensive. The Taguchi method of design of experiments is a simple statistical tool involving a system of tabulated designs (arrays) that allows a maximum number of main effects to be estimated in an unbiased (orthogonal) fashion with a minimum number of experimental runs. It has been applied to predict the significant contribution of the design variable(s) and the optimum combination of each variable by conducting experiments on a real-time basis. The modeling that is performed essentially relates signal-to-noise ratio to the control variables in a 'main effect only' approach. This approach enables both multiple response and dynamic problems to be studied by handling noise factors. Taguchi principles and concepts have made extensive contributions to industry by bringing focused awareness to robustness, noise and quality. This methodology has been widely applied in many industrial sectors; however, its application in biological sciences has been limited. In the present review, the application and comparison of the Taguchi methodology has been emphasized with specific case studies in the field of biotechnology, particularly in diverse areas like fermentation, food processing, molecular biology, wastewater treatment and bioremediation.

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Untitled

Cited by 9 publications

References 9 publications

Influence of induction conditions on the expression of carbazole dioxygenase components (CarAa, CarAc, and CarAd) from Pseudomonas stutzeri in recombinant Escherichia coli using experimental design

Influence of induction conditions on the expression of carbazole dioxygenase components (CarAa, CarAc, and CarAd) from Pseudomonas stutzeri in recombinant Escherichia coli using experimental design

The Goldilocks Approach: A Review of Employing Design of Experiments in Prokaryotic Recombinant Protein Production

The Taguchi methodology as a statistical tool for biotechnological applications: A critical appraisal

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