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Prodigiosin (Pg), a secondary metabolite with anticancer and antimicrobial activities, can be produced in Serratia marcescens bacteria through the condensation reaction of 4-methoxy-2, 2’-bipyrrole-5-carboxyaldehyde (MBC) and 2-methyl-3-amylpyrrole (MAP). Among these, the MBC synthetic pathway is started by the conversion of L-proline to L-proline-AMP before this complex is covalently attached to PigG. This reaction is catalyzed by an L-prolyl-AMP ligase named PigI. Therefore, PigI protein plays an important role in the prodigiosin biosynthetic pathway. However, studies related to PigI protein have not been carried out in Vietnam yet. In this work, the pigI gene was cloned and expressed in Escherichia coli DH10B and BL21 (DE3), respectively. Sequence alignment results revealed that the obtained pigI gene is 99.7% identical to the four strains, CP027798, CP027796, CP021984 and CP003959. This recombinant vector pJET1.2/pigI was used to reamplify pigI, and the acquired amplicon was inserted into pET22b vector at the site of HindIII and XhoI. The clone E. coli BL21 (DE3) containing the recombinant vector pET22b/pigI was expressed in an auto-induced medium. The presence of PigI protein in the lysate was identified due to a 53 kDa band through Western Blot analysis using an anti-his-tag antibody. The results of our study provide a potential method for producing prodigiosin from recombinant protein in Vietnam.

In many industrial sectors continuous processing is already the golden standard to maximize productivity. However, when working with living cells, subpopulation formation causes instabilities in long-term cultivations. In cascaded continuous cultivation, biomass formation and recombinant protein expression can be spatially separated. This cultivation mode was found to facilitate stable protein expression using microbial hosts, however mechanistic knowledge of this cultivation strategy is scarce. In this contribution we present a method workflow to reduce workload and accelerate the establishment of stable continuous processes with E. coli BL21(DE3) exclusively based on bioengineering methods.

There is increased food industry attention to low-calorie natural sweeteners with good taste properties such as in plant-based sweet proteins. Brazzein is an attractive alternative to sucrose becau...

Objective: The objective of this study was to evaluate two signal peptides (TorA and PelB), representing the most common secretion pathways in Escherichia coli, for their ability to secrete recombinant human epidermal growth factor (rhEGF) protein in the extracellular expression. Methods: E. coli BL21 (DE3) as the host cell to be transformed using recombinant plasmid pD881-TorA the consensus already containing hEGF gene and the signal peptide TorA or PelB, then expressed by L-rhamnose induction. rhEGF purified by heat treatment and ion-exchange chromatography. The hEGF protein was characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ELISA. Results: The result showed that PelB was secreting more hEGF protein compared to TorA with protein expression results of 48.2 μg/L and purification results of 0.360 μg/L, with a purity level of 83%. Conclusion: The results of this study explain in extracellular expression of hEGF protein in E. coli, PelB helps hEGF protein secretion to culture media better than TorA.

Avoiding acetate accumulation is a major challenge in Escherichia coli fed-batch cultures, since it leads to an inhibition of the cell respiratory capacity. A closed-loop regulation ensuring a low acetate concentration offers a practical solution to maintain the cultures near the optimal operating conditions. In this work, a robust Generic Model Controller (GMC) is designed and implemented to regulate the acetate concentration in E. coli BL21 (DE3) fed-batch cultures. To compensate for model mismatch, disturbances, and measurement noise, a robust design using the L M I formalism is achieved, considering robustness and transient performance requirements. Since the acetate concentration is not available for on-line measurement, an Unscented Kalman Filter (UKF) is also designed and implemented to estimate the acetate concentration based on an on-line biomass measurement. The proposed GMC-UKF strategy is validated through simulation runs and experimental tests at lab-scale. The control strategy shows good performance in regulating the non-measured acetate concentration. The estimation of this latter signal from the biomass measurement is performed accurately by the UKF.

The impact of recombinant protein production on carbon and energy metabolism in Escherichia coli BL21 (DE3) was studied through transcriptome and proteome analysis of cells induced in carbon‐limited fed‐batch cultures during either fast or slow growth. Production of human basic fibroblast growth factor (pET expression system, T7 promoter) during fast growth leads to a macroscopically observable response classifiable into two consecutive steps: i. apparently unperturbed growth and respiration with concomitant formation of pyruvate and acetate followed by ii. inhibition of growth, respiratory activity and glucose uptake. Down‐regulation of genes involved in sugar and acetate uptake, tricarboxylic acid (TCA) cycle, and respiratory energy generation started already during apparently unperturbed growth with the exceptions of up‐regulated genes encoding the less energy efficient NADH dehydrogenase and terminal oxidases. A transcription factor target gene network analysis revealed that observed changes are mainly attributable to the vanishing influence of the transcription factor CRP‐cAMP but also to a strong down‐regulation of AcrA‐P repressed genes. Moreover, down‐regulation of MalT activated and up‐regulation of PdhR repressed genes contribute among others to the reorganization of the transcriptome. The main drivers were identified as accumulating metabolites, for example, pyruvate, which affect transcription factor activity. The resulting restructured proteome leads to reduced glucose uptake, TCA cycle, and respiratory capacities this way decreasing catabolic carbon breakdown and metabolite accumulation. At slow growth, the production provoked transcriptome rearrangements are more subtle not leading to a macroscopically evident response. In summary, the transcriptomic response towards recombinant gene expression mimics a carbon or nutrient up‐shift response aiming to match catabolic carbon processing with compromised anabolic capacities of induced cells. It is not the reason for growth inhibition and the metabolic burden but the cellular attempt to attenuate the “toxic effect” of recombinant gene expression by reducing carbon catabolism.

Continuous processing allows to maximize space-time yields and is implemented in many industrial branches. However, in manufacturing of value added compounds produced with microbial hosts, continuous processing is not state-of-the-art yet. This is because fluctuating productivity causes unwanted process deviations. Cascaded continuous bioprocessing, unlike conventional continuous process modes, was found to result in stable productivity. This manuscript serves as a guideline how to set up a cascaded continuous cultivation with Escherichia coli BL21 DE(3).

Objective: To demonstrate the sensitivity of expression of fusion genes to existence of a large number of rare codons in recombinant gene sequenced. Methods: Primers for amplification of cholera toxin B, Shiga toxin B and gfp genes were designed by Primer3 software and synthesized. All of these 3 genes were cloned. Then the genes were fused together by restriction sites and enzymatic method. Two linkers were used as a flexible bridge in connection of these genes. Results: Cloning and fusion of cholera toxin B, Shiga toxin B and gfp genes were done correctly. After that, expression of the recombinant gene construction was surveyed. Conclusions: According to what was seen, because of the accumulation of 12 rare codons of Shiga toxin B and 19 rare codons of cholera toxin B in this gene cassette, the expression of the recombinant gene cassette, in Escherichia coli BL21, failed.

TNF-α, a prototype member of the TNF family of ligands, has both pro-inflammatory and immune-regulatory functions, which make it as an appropriate therapeutic target for selective blockade in antibody therapy of many diseases like in rheumatoid arthritis. Using two models of SHuffle® T7 Express and BL21 (DE3) cells, we have expressed this protein and recognized it by SDS-PAGE analysis. FTIR biospectroscopy of the resulted purified proteins has been performed and mathematical calculations has been done for further identification of the structural and conformational differences between the two products. Our results showed some differences in disulfide bond formation and β-sheet turns between these two recombinant proteins. To the best of our knowledge, this is the first study that compare secondary structure of recombinant proteins in both conventional and next generation Escherichia coli based expression systems using reliable, simple, rapid and economic ATR-FTIR analysis. Whether these differences might have significant effects on TNF-α inflammatory and immune-regulatory function in biological systems might be very much important and need further investigations.

The dynamin-related proteins (DRPs) are self-assembling membrane remodeling machines that are indispensable for fundamental cellular trafficking and homeostatic processes. We describe in this chapter protocols developed in our laboratory for purification of full-length and minimal constructs of Chaetomium thermophilum Vps1, the model fungal DRP, using mammalian and Escherichia coli expression systems.

Many proteins produced in E. coli accumulate in inclusion bodies. This study aims to detect the role of temperature in reducing the formation of inclusion bodies during recombinant human prethrombin-2expressed in E. coli BL21 (DE3) Arctic Express host. In this study, we created a host growth curve to find out the right time to add an inducer. The inducer used in our experiment was IPTG 0.1 mM. The fermentation process use a temperature of 12°C and 22°C. The results showed that recombinant human prethrombin-2 was successfully expressed as protein soluble using an optimum temperature of 12°C in E. coli BL21 (DE3) Arctic Express. It was indicated from the 63kDa protein band obtained from the soluble fraction on SDS-PAGE. The higher temperature of fermentation increased the amount of protein in the insoluble fraction due. It concluded that the fermentation temperature affect the rate of prethrombin-2 expression. Keywords: E. coli BL21(DE3) ArcticExpress, prethrombin-2, soluble, temperature

The impact of cultivation strategy on the cost of recombinant protein production is crucial for defining cost-effective bioreactor operation conditions. This paper presents a methodology to estimate and compare cost impacts related to utilities as well as medium composition, using simple design equations and accessible data. Data from batch bioreactor cultures were used as case study involving the production of pneumococcal surface protein A, a soluble recombinant protein, employing E. coli BL21(DE3). Cultivation strategies and corresponding process costs covered a wide range of operational conditions, including different media, inducers, and temperatures. The core expenses were related to the medium and cooling. When the price of peptone was above the threshold value of US$ 30/kg, defined medium became the best choice. IPTG and temperatures around 32 °C led to shorter cultures and lower PspA4Pro production costs. The procedure offers a simple, accessible theoretical tool to identify cost-effective production strategies using bioreactors.

Production D-xylonic acid from recombinant E. coli BL21 (DE3) at a small scale has been successfully done several times. In an attempt to scale up the production process to a bigger scale, fermentation in bioreactor was conducted. The comparison on growth of recombinant E. coli BL21 (DE3) and production of D-xylonic acid in both shake flask and bioreactor was investigated. Higher specific growth rate of recombinant E. coli BL21 (DE3) and concentration of D-xylonic acid however, was obtained by fermentation in shake flask which is 0.273 h−1 and 9.06 gL−1 D-xylonic acid respectively. The result suggests several mistakes done during fermentation in bioreactor that causes low concentration of D-xylonic acid in bioreactor fermentation. Therefore, this calls up for improvement and better scale-up strategies in future work.

l-isoleucine dioxygenase (IDO) is a kind of Fe(II)/α-ketoglutarate (α-KG)-dependent hydroxylase, with the ability of converting l-isoleucine to (2S,3R,4S)-4-hydroxyisoleucine (4-HIL). IDO encoding gene ido from Bucillus thuringiensis TCCC 11826 was cloned into Escherich coli BL21(DE3) to express IDO. To optimized the condition for recombinant IDO expression in E. coli BL21(DE3), the effect of isopropyl-β-d-thiogalactoside (IPTG) concentration, induction temperature, induction time and expression time on expression were studied. E. coli BL21/pEThis-ido was successfully constructed and the amount of expressed protein was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDA-PAGE). Results showed that induction for 6 h with 0.15 mmol/L IPTG after recombinant E. coli BL21(DE3) cultured for 4 h at 37 °C lead to the highest recombinant IDO level. This work will lay basis on the microbial expression technology of IDO and Fe(II)/(α-KG)-dependent hydroxylase.

Applying Escherichia coli (E. coli) for fermentation is a very common technology. However combined with genetic engineering techniques to construct the recombinant Escherichia coli and study their growth characteristics has become the hot spot now. The recombinant Escherichia coli BL21 (DE3) /pET-28baroGM150 had been constructed by our laboratory in the previous experiment. And the purpose of this study was to optimize the proliferation conditions of Escherichia coli BL21 (DE3) /pET-28baroGM150. In order to make the recombinant Escherichia coli grow stable under suitable conditions, using the density of bacteria and plasmid stability as indexes, three factors were tested including temperature, initial pH and loading volume. And the results indicated that the optimal proliferation temperature of the recombinant strain was 30°C, initial pH value was 6.5, loading volume was 150 mL medium of 1000 mL bottles.

When producing recombinant proteins, the use of Escherichia coli strain BL21(DE3) in combination with the T7-based pET-expression system is often the method of choice. In a recent study we introduced a mechanistic model describing the correlation of the specific glucose uptake rate (qs,glu) and the corresponding maximum specific lactose uptake rate (qs,lac,max) for a pET-based E. coli BL21(DE3) strain producing a single chain variable fragment (scFv). We showed the effect of qs,lac,max on productivity and product location underlining its importance for recombinant protein production. In the present study we investigated the mechanistic qs,glu/qs,lac,max correlation for four pET-based E. coli BL21(DE3) strains producing different recombinant products and thereby proved the mechanistic model to be platform knowledge for E. coli BL21(DE3). However, we found that the model parameters strongly depended on the recombinant product. Driven by this observation we tested different dynamic bioprocess strategies to allow a faster investigation of this mechanistic correlation. In fact, we succeeded and propose an experimental strategy comprising only one batch cultivation, one fed-batch cultivation as well as one dynamic experiment, to reliably determine the mechanistic model for qs,glu/qs,lac,max and get trustworthy model parameters for pET-based E. coli BL21(DE3) strains which are the basis for bioprocess development.

Production of recombinant proteins in E. coli still facesa bottle neck due to formation of inclusion bodies. The level of gene transcription can be regulated by using appropriate concentration of isopropyl-β-D-thiogalactoside (IPTG) inducer. The purpose of this study is to determine the effect of IPTG concentration on human prethrombin-2 (hPT-2) expression. The hPT-2 expression was induced by various concentrations of IPTG (0.01 mM, 0.025 mM, 0.05 mM, 0.075 mM, 0.1 mM, 0.2 mM and 0.3 mM) at 12°C for 18 hours hosted by E. coli BL21(DE3) ArcticExpress. The result show that the suitable IPTG concentration for induction of hPT-2 in E. coli BL21(DE3) ArcticExpress was 0.1 mM. It was indicated from the 62-kDa protein band obtained from the soluble fraction on SDS-PAGE. It concluded that the IPTG concentration affect the rate of rhPT-2 expression.

EMAP II (Endothelial Monocyte-Activating Polypeptide) – a new antiangiogenic proinflammatory cytokine that exhibits antitumor activity. In order to develop genetically engineered technology for EMAP II optimization of bacterial expression conditions within pET30a vector encoded EMAP II was carried out. Both the influence of target protein synthesis inductor IPTG concentration on its overall yield and optimal bacterial cultivation time before and after inductor addition were estimated. There was a proposed scheme for the cultivation of culture E. colі BL21(DE3)pLysE to achieve high yield of recombinant cytokine EMAP II at the level of 110 mg from 1 liter of bacterial culture.

Antecedents of Escherichia coli B have been traced through publications, inferences, and personal communication to a strain from the Institut Pasteur in Paris used by d'Herelle in his studies of bacteriophages as early as 1918 (a strain not in the current collection). This strain appears to have passed from d'Herelle to Bordet in 1920, and from Bordet to at least three other laboratories by 1925. The strain that Gratia received from Bordet was apparently passed to Bronfenbrenner by 1924 and from him to Luria around 1941. Delbrück and Luria published the first paper calling this strain B in 1942. Its choice as the common host for phages T1-T7 by the phage group that developed around Delbrück, Luria, and Hershey in the 1940s led to widespread use of B along with E. coli K-12, chosen about the same time for biochemical and genetic studies by Tatum and Lederberg. Not all currently available strains related to B are descended from the B of Delbrück and Luria; at least three strains with somewhat different characteristics were derived independently by Hershey directly from the Bronfenbrenner strain, and a strain that appears to have passed from Bordet to Wollman is in the current Collection of the Institut Pasteur. The succession of manipulations and strains that led from the B of Delbrück and Luria to REL606 and BL21(DE3) is given, established in part through evidence from their recently determined complete genome sequences.

The optimization of the conditions of bacterial expression of recombinant tyrosyl-tRNA synthetase was conducted. The influence of the concentration of synthesis inductor of target protein at its final output and the best time of cultivation of bacterial culture before and after inductor adding was investigated. The optimal conditions for culture E. coli BL21 (DE3) pLysE cultivation to achieve a high expression level of recombinant protein were proposed.

To eliminate unavoidable contamination of purified recombinant proteins by DnaK, we present a unique approach employing a BL21(DE3) Δ dnaK strain of Escherichia coli . Selected representative purified proteins remained soluble, correctly assembled, and active. This finding establishes DnaK dispensability for protein production in BL21(DE3), which is void of Lon protease, key to eliminating unfolded proteins.

The ability of catalyzing indole into indigo of gene engineering strain expressing P450 BM3 immobilized by entrapment in calcium-alginate gel capsules was examined, and various characteristics of immobilized cells were assessed. Optimum conditions for cells activity were not affected after immobilization, and pH and temperature for both free and immobilized cells were found to be pH 7.5 and 35°C, respectively. The immobilized cells exhibited a markedly improved thermal stability than free cells. After five repeated experiments, the yield of indigo with the immobilized cells retained over 94% of their original activity, which indicated that the operational stability for recycling in batch processes was improved.

Physical optimization is important for enzyme production by fermentation process. In general, fermentation process at optimal condition increases the expression and production level of enzyme to many times in comparison with their natural production. This study was focused on the optimization of the physical factors that influenced the thermostable alkaline protease production. The induction and incubation time were studied using conventional method while the other three factors which are incubation temperature, initial pH of medium and agitation speed were optimized by response surface methodology (RSM). The interaction effects among these factors were explained using response plot and the model adequacy was satisfactory as the coefficient of determination (R2) was 96.48%. The enhancement of thermostable protease from 197.83 U/ml to 325.89 U/ml was achieved using both conventional and statistical approach of response surface methodology (RSM). This present study proved that physical optimization significantly affects the protease production and the optimum physical condition obtained may applied in large scale process.

To characterize a novel feruloyl esterase from Escherichia coli BL21 DE3. The gene encoding BioH was cloned and overexpressed in E. coli. The protein was purified and its catalytic activity was assessed. BioH exhibited feruloyl esterase activity toward a broad range of substrates, and the corresponding kinetic constants for the methyl ferulate, ethyl ferulate, and methyl p-coumarate substrates were: K m values of 0.48, 6.3, and 1.9 mM, respectively, and k cat /K m values of 9.3, 3.8, and 3.8 mM−1 s−1, respectively. Feruloyl esterase from E. coli was expressed for the first time. BioH was confirmed to be a feruloyl esterase.

This is the protocol for Protein Expression Using BL21(DE3) Competent E. coli (C2527). For large scale, please follow this protocol variant.

This is the protocol for a large-scale Protein Expression Using BL21(DE3) Competent E. coli cells(C2527).

A control structure allowing all varying process parameters to be utilised in highlevel control decisions was developed and implemented in a 5 litre reactor system. The integrated control structure was used to implement PI control of OUR/CER ratio (1/RQ) in a fed batch fermentation of E.coli BL21 DE3 on complex media. The feed controller was successful, with setpoints between 1.3 and 1.5 maintained. Future development of the integrated control system is discussed.

His-tag affinity purification is one of the most commonly used methods to purify recombinant proteins expressed in E. coli. One drawback of using the His-tag is the co-purification of contaminating histidine-rich E. coli proteins. We engineered a new E. coli expression strain, LOBSTR (low background strain), which eliminates the most abundant contaminants. LOBSTR is derived from the E. coli BL21(DE3) strain and carries genomically modified copies of arnA and slyD, whose protein products exhibit reduced affinities to Ni and Co resins, resulting in a much higher purity of the target protein. The use of LOBSTR enables the pursuit of challenging low-expressing protein targets by reducing background contamination with no additional purification steps, materials, or costs, and thus pushes the limits of standard His-tag purifications., Lundbeck Foundation, Danish Council for Independent Research (Postdoctoral Grant), National Institutes of Health (U.S.) (Pew Scholar Award Grant GM077537)

Effects of oxygen transfer (OT) on benzaldehyde lyase (BAL) production by recombinant E. coli BL21 (DE3) pLySs were investigated at an air-inlet rate of Q(O)/V(R) = 0.5 vvm and agitation rates of N = 250, 500, 625, and 750/min; and at constant dissolved oxygen (DO) concentrations of C(DO) = 0.04 and 0.08 mol/m(3), on a glucose based defined medium. The highest cell concentration (C(X) = 3.0 kg/m(3)) and volumetric BAL activity (A = 1095 U/cm(3)) were obtained at C(DO) = 0.08 mol/m(3). K(L)a increased with agitation rate and decreased with C(X). The highest K(L)a (= 0.039 s((-1)) was obtained at Q(O)/V(R) = 0.5 vvm and N = 750/min. Damkohler number increased with decrease in agitation rate and increase in C(X), while the effectiveness factor was inversely proportional to C(X). The specific growth rate and the yield coefficients decreased with cultivation time and higher values were obtained in higher agitation rates. Oxygen consumption and glucose consumption for maintenance were changed proportionally and inverse proportionally with the BAL production, respectively.

The T7 polymerase-based pET System™ is one of the most powerful and widely used prokaryotic expression systems available today. Expression of even slightly toxic gene products in BL21 (DE3), however, has been problematic due to basal expression, which leads to decreased plasmid stability and variable yields following large-scale growth and induction. Use of host strains such as BL21 (DE3) pLysS provides high stringency and consistent expression but typically at the cost of reduced protein levels upon induction. The experiments presented here suggest that catabolite repression can effectively reduce basal expression of the T7 polymerase gene in BL21 (DE3), yielding tight regulation and consistency comparable to that of BL21 (DE3) pLysS. By switching to a poor carbon source for the final growth cycles, the higher expression levels typical of BL21 (DE3) can readily be obtained upon induction.