scholarly journals Optimization of culture conditions for the expression of three different insoluble proteins in Escherichia coli

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Matías Gutiérrez-González ◽  
Camila Farías ◽  
Samantha Tello ◽  
Diana Pérez-Etcheverry ◽  
Alfonso Romero ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Induction Time ◽  
Recombinant Protein Expression ◽  
Principal Component ◽  
Culture Conditions ◽  
Binding Assays ◽  
Iptg Concentration ◽  
Post Induction ◽  
Inclusion Body Formation ◽  
Induction Temperature

AbstractRecombinant protein expression for structural and therapeutic applications requires the use of systems with high expression yields. Escherichia coli is considered the workhorse for this purpose, given its fast growth rate and feasible manipulation. However, bacterial inclusion body formation remains a challenge for further protein purification. We analyzed and optimized the expression conditions for three different proteins: an anti-MICA scFv, MICA, and p19 subunit of IL-23. We used a response surface methodology based on a three-level Box-Behnken design, which included three factors: post-induction temperature, post-induction time and IPTG concentration. Comparing this information with soluble protein data in a principal component analysis revealed that insoluble and soluble proteins have different optimal conditions for post-induction temperature, post-induction time, IPTG concentration and in amino acid sequence features. Finally, we optimized the refolding conditions of the least expressed protein, anti-MICA scFv, using a fast dilution protocol with different additives, obtaining soluble and active scFv for binding assays. These results allowed us to obtain higher yields of proteins expressed in inclusion bodies. Further studies using the system proposed in this study may lead to the identification of optimal environmental factors for a given protein sequence, favoring the acceleration of bioprocess development and structural studies.

Recombinant Protein Expression in Escherichia coli (E.coli): What We Need to Know

2018 ◽  
Vol 24 (6) ◽  
pp. 718-725 ◽  
Author(s):  
Seyed Mohammad Gheibi Hayat ◽  
Najmeh Farahani ◽  
Behrouz Golichenari ◽  
Amirhossein Sahebkar
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Proteins ◽  
Recombinant Protein Expression ◽  
Culture Conditions ◽  
Strain Vector ◽  
Factors Affecting ◽  
Expression In Escherichia Coli ◽  
Pros And Cons

Background: Host, vector, and culture conditions (including cultivation media) are considered among the three main elements contributing to a successful production of recombinant proteins. Accordingly, one of the most common hosts to produce recombinant therapeutic proteins is Escherichia coli. Methodology: A comprehensive literature review was performed to identify important factors affecting production of recombinant proteins in Escherichia coli. Results: Escherichia coli is taken into account as the easiest, quickest, and cheapest host with a fully known genome. Thus, numerous modifications have been carried out on Escherichia coli to optimize it as a good candidate for protein expression and; as a result, several engineered strains of Escherichia coli have been designed. In general; host strain, vector, and cultivation parameters are recognized as crucial ones determining success of recombinant protein expression in Escherichia coli. In this review, the role of host, vector, and culture conditions along with current pros and cons of different types of these factors leading to success of recombinant protein expression in Escherichia coli were discussed. Conclusion: Successful protein expression in Escherichia coli necessitates a broad knowledge about physicochemical properties of recombinant proteins, selection among common strains of Escherichia coli and vectors, as well as factors related to media including time, temperature, and inducer.

Excretory overexpression of Paenibacillus pabuli US132 cyclodextrin glucanotransferase (CGTase) in Escherichia coli: gene cloning and optimization of the culture conditions using experimental design

Biologia ◽  
2011 ◽  
Vol 66 (6) ◽  
Author(s):  
Dorra Ayadi ◽  
Radhouane Kammoun ◽  
Sonia Jemli ◽  
Samir Bejar
Keyword(s):  
Escherichia Coli ◽  
Bacillus Stearothermophilus ◽  
Active Form ◽  
Culture Conditions ◽  
Cyclodextrin Glucanotransferase ◽  
Gene Encoding ◽  
Box Behnken Design ◽  
Post Induction ◽  
Optimized Conditions ◽  
Maximum Expression

AbstractThe gene encoding the cyclodextrin glucanotransferase of Paenibacillus pabuli US132 was connected to the amylase signal peptide of Bacillus stearothermophilus. This leads to an efficient secretion of the recombinant enzyme into the culture medium of Escherichia coli as an active form contrasting with the native construction leading to a periplasmic production. The optimum cultivation conditions for the maximum expression were optimized, using a Box-Behnken design under the response surface methodology, and found to be a post-induction temperature of 24°C, an induction-starting A600 nm of 0.85, an isopropyl-β-D-thiogalactopyranoside level of 0.045 mM and a post-induction time of 3.9 h. The screening of media components and their concentration were achieved using a Plackett-Burman and a Box-Behnken designs sequentially. Under the optimized conditions selected and in agreement with the predicted model, an activity of 6.03 U/mL was attained. This CGTase production was three-times higher than that using the non-optimized culture conditions (2 U/mL).

Conditions Resulting in Formation of Properly Assembled Retroviral Capsids within Inclusion Bodies of Escherichia coli

1999 ◽  
Vol 64 (8) ◽  
pp. 1348-1356 ◽  
Author(s):  
Michaela Rumlová-Kliková ◽  
Iva Pichová ◽  
Eric Hunter ◽  
Tomáš Ruml
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Amorphous Material ◽  
Bacterial Expression ◽  
Biologically Active ◽  
Capsid Assembly ◽  
Cultivation Medium ◽  
Viral Particles ◽  
Induction Temperature

It has been generally accepted that inclusion bodies (IBs) formed in Escherichia coli consist of non-biologically active aggregated proteins, which are stabilized by non-productive interactions. We show here that bacterial expression of a retroviral capsid polyprotein results in formation of insoluble IBs that contain fully assembled viral particles connected with amorphous material. The efficiency of IBs formation and capsid assembly was not significantly affected by changes in induction temperature, pH of cultivation medium or the level of expression.

Finite Element Model to Predict the Bioconversion Rate of Glucose to Fructose using Escherichia coli K12 for Sugar Production from Date

2015 ◽  
Vol 11 (1) ◽  
pp. 105-113 ◽  
Author(s):  
Maher Trigui ◽  
Karim Gabsi ◽  
Walid Zneti ◽  
Suzelle Barrington ◽  
Ahmed Noureddine Helal
Keyword(s):  
Escherichia Coli ◽  
Induction Time ◽  
Volume Fraction ◽  
Element Model ◽  
Initial Concentration ◽  
Computational Fluids Dynamics ◽  
Date Syrup ◽  
Computational Fluids ◽  
Two Phases ◽  
Inoculum Volume

Abstract In this study, Bioconversion process of glucose to fructose from date syrup using Escherichia coli K12 is modeled using a commercial computational fluids dynamics (CFD) code fluent FLUENT 6.3.23 [8] which we implemented a user-defined functions (UDF) to simulate the interrelationships at play between various phases. A two phases CFD model was developed using an Eulerian – Eulerian approach to calculate the fructose volume fraction produced during time. The bioconversion process was studied as function of three initial concentration of glucose (0.14, 0.242 and 0.463gL–1), three induction time (60, 120 and 180 mn) and three inoculum volume (100, 120 and 150mL). The numerical results are compared with experimental data for bioconversion rate and show good agreement (R2= 0.894). The optimal condition of diffusion was obtained by applying an initial concentration of glucose less than 0.2gL–1 and induction time great than 100 minutes.

Optimized Culture Conditions for the Efficient Production of Porcine Adenylate Kinase in Recombinant Escherichia coli

2010 ◽  
Vol 162 (3) ◽  
pp. 823-829 ◽  
Author(s):  
Toru Matsui ◽  
Takashi Togari ◽  
Satoru Misawa ◽  
Tomoyuki Namihira ◽  
Naoya Shinzato ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Adenylate Kinase ◽  
Culture Conditions ◽  
Recombinant Escherichia Coli ◽  
Efficient Production

Study on Optimization of Fermentation Condition for Nitrilase-Producer Escherichia Coli BL21 (DE3)/pET-Nit

2011 ◽  
Vol 175-176 ◽  
pp. 192-196 ◽  
Author(s):  
Li Li Feng ◽  
Jian Fei Zhang ◽  
Hui Luo ◽  
Zheng Li ◽  
Hong Jie Zhang
Keyword(s):  
Escherichia Coli ◽  
Recombinant Strain ◽  
Culture Medium ◽  
Culture Conditions ◽  
Fermentation Condition ◽  
Hydrogen Phosphate ◽  
Strain Bl21 ◽  
Initial Ph ◽  
Potassium Hydrogen ◽  
Optimization Of Fermentation

The paper concentrated on the optimization of the recombinant strain BL21 (DE3)-PE7-Nit. The component of culture medium and the culture conditions were optimized. The optimized medium was: yeast extract 10 g/l, L-glutamate sodium 8 g/l, MgSO4.7H2O 0.7 g/l, Isopropyl-β-D-thiogalactopyranoside 0.3 mmol/L, potassium hydrogen phosphate 0.5 g / L, phosphate Potassium 0.5 g / L and the culture condition was: initial pH 7.0, inoculum 2%. The result showed that the activity of nitrilase prepared with these conditions increased by 130.37 % through optimization.

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