Production of a new sea anemone neurotoxin by recombinant Escherichia coli: Optimization of culture conditions using response surface methodology

Wang, Yonghua; Jing, Chenfeng; Yang, Bo; Mainda, Geoffrey; Dong, Meiling; Xu, Anlong

doi:10.1016/j.procbio.2004.12.024

Cited by 29 publications

(28 citation statements)

References 21 publications

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“…2. The equation was very reliable with R 2 value of 0.9365 and low p values (p \ 0.1) of the model fit, which indicate a strong correspondence between theoretical predictions and experimental results [11,12]. The low coefficient of variation (CV) value of 7.956054 also represented that the differences between the predicted and the observed values were little.…”

Section: Pbd and Analysismentioning

confidence: 72%

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Optimization of culture medium for enhanced production of exopolysaccharide from Aureobasidium pullulans

Yoon

Hong

Kim

et al. 2011

Bioprocess Biosyst Eng

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Polysaccharides produced by microorganisms are utilized for a variety of purposes, including the use in cosmetics and as food additives. More recently, polysaccharides have been exploited by the medical and pharmaceutical industries, and those originated from many species of mushrooms have been especially useful in industrial applications; however, the production and synthesis of these compounds is costly and time consuming. In this study, we developed a method for low-cost production of exopolysaccharide (EPS) that effectively screens components and optimizes medium composition using statistical methods (Plackett-Burman and Box-Behnken design). As a result, we obtained the following optimized medium: sucrose 165.73 g/L, sodium nitrate 3.08 g/L, dipotassium phosphate 1.00 g/L, potassium chloride 0.50 g/L, magnesium sulfate 0.50 g/L, ferrous sulfate 0.01 g/L, and 0.71 g/L of Ashbya gossypii extract. The maximum production of about 29 g/L EPS was achieved in the optimized medium during 84 h batch fermentation.

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Section: Pbd and Analysismentioning

confidence: 72%

“…Recently efforts were made to screen a cost-effective nutrient [10] or to optimize culture media [11] for the EPS production from A. pullulans. However, such traditional optimization strategies (one-factor-at-a-time) require considerable time and effort [12].…”

Section: Introductionmentioning

confidence: 99%

Optimization of culture medium for enhanced production of exopolysaccharide from Aureobasidium pullulans

Yoon

Hong

Kim

et al. 2011

Bioprocess Biosyst Eng

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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%

“…It is discussed in the literature that recombinant protein production in E. coli is proportional to the specific growth rate at induction if cell metabolic capacity is at its best condition [33]. In view of this observation, induction is usually performed at the exponential phase, but depending on the protein and induction system [9,19,30,32,47,53], protein expression can vary with a lower or higher cell concentration prior to induction (i.e., early or late logarithmic phase). In many cases, the best approach is to maximize the number of viable cells prior to the addition of the inducer, which often retards cell growth [19].…”

Section: Discussionmentioning

confidence: 99%

“…Temperature is related to modulations in chaperone activity or other accessory proteins involved in protein folding [51], so it is associated with proteins obtained in soluble form, which are useful for further purification [19,49]. Temperature is also reported to affect plasmid stability in recombinant E. coli cultures [45,53]. Cultivation at lower temperatures can reduce protein aggregation into inclusion bodies, proteolytic degradation, and responses to cell stress conditions, leading to a higher production of soluble recombinant proteins [10,30,31,52,53].…”

Section: Discussionmentioning

confidence: 99%

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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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Framework for the Rapid Optimization of Soluble Protein Expression inEscherichia coliCombining Microscale Experiments and Statistical Experimental Design

Islam¹,

Tisi²,

Levy³

et al. 2007

Biotechnol Progress

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A major bottleneck in drug discovery is the production of soluble human recombinant protein in sufficient quantities for analysis. This problem is compounded by the complex relationship between protein yield and the large number of variables which affect it. Here, we describe a generic framework for the rapid identification and optimization of factors affecting soluble protein yield in microwell plate fermentations as a prelude to the predictive and reliable scaleup of optimized culture conditions. Recombinant expression of firefly luciferase in Escherichia coli was used as a model system. Two rounds of statistical design of experiments (DoE) were employed to first screen (D-optimal design) and then optimize (central composite face design) the yield of soluble protein. Biological variables from the initial screening experiments included medium type and growth and induction conditions. To provide insight into the impact of the engineering environment on cell growth and expression, plate geometry, shaking speed, and liquid fill volume were included as factors since these strongly influence oxygen transfer into the wells. Compared to standard reference conditions, both the screening and optimization designs gave up to 3-fold increases in the soluble protein yield, i.e., a 9-fold increase overall. In general the highest protein yields were obtained when cells were induced at a relatively low biomass concentration and then allowed to grow slowly up to a high final biomass concentration, >8 g.L-1. Consideration and analysis of the model results showed 6 of the original 10 variables to be important at the screening stage and 3 after optimization. The latter included the microwell plate shaking speeds pre- and postinduction, indicating the importance of oxygen transfer into the microwells and identifying this as a critical parameter for subsequent scale translation studies. The optimization process, also known as response surface methodology (RSM), predicted there to be a distinct optimum set of conditions for protein expression which could be verified experimentally. This work provides a generic approach to protein expression optimization in which both biological and engineering variables are investigated from the initial screening stage. The application of DoE reduces the total number of experiments needed to be performed, while experimentation at the microwell scale increases experimental throughput and reduces cost.

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Production of a new sea anemone neurotoxin by recombinant Escherichia coli: Optimization of culture conditions using response surface methodology

Cited by 29 publications

References 21 publications

Optimization of culture medium for enhanced production of exopolysaccharide from Aureobasidium pullulans

Optimization of culture medium for enhanced production of exopolysaccharide from Aureobasidium pullulans

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

Framework for the Rapid Optimization of Soluble Protein Expression inEscherichia coliCombining Microscale Experiments and Statistical Experimental Design

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