scholarly journals Regulating the T7 RNA polymerase expression in E. coli BL21 (DE3) to provide more host options for recombinant protein production

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Fei Du ◽  
Yun-Qi Liu ◽  
Ying-Shuang Xu ◽  
Zi-Jia Li ◽  
Yu-Zhou Wang ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Rna Polymerase ◽  
Recombinant Proteins ◽  
Expression System ◽  
T7 Rna Polymerase ◽  
Expression Level ◽  
Foreign Protein ◽  
Atpase Subunit ◽  
E Coli ◽  
Prokaryotic Expression System

AbstractEscherichia coli is the most widely used bacterium in prokaryotic expression system for the production of recombinant proteins. In BL21 (DE3), the gene encoding the T7 RNA polymerase (T7 RNAP) is under control of the strong lacUV5 promoter (PlacUV5), which is leakier and more active than wild-type lac promoter (PlacWT) under certain growth conditions. These characteristics are not advantageous for the production of those recombinant proteins with toxic or growth-burdened. On the one hand, leakage expression of T7 RNAP leads to rapid production of target proteins under non-inducing period, which sucks resources away from cellular growth. Moreover, in non-inducing or inducing period, high expression of T7 RNAP production leads to the high-production of hard-to-express proteins, which may all lead to loss of the expression plasmid or the occurrence of mutations in the expressed gene. Therefore, more BL21 (DE3)-derived variant strains with rigorous expression and different expression level of T7 RNAP should be developed. Hence, we replaced PlacUV5 with other inducible promoters respectively, including arabinose promoter (ParaBAD), rhamnose promoter (PrhaBAD), tetracycline promoter (Ptet), in order to optimize the production of recombinant protein by regulating the transcription level and the leakage level of T7 RNAP. Compared with BL21 (DE3), the constructed engineered strains had higher sensitivity to inducers, among which rhamnose and tetracycline promoters had the lowest leakage ability. In the production of glucose dehydrogenase (GDH), a protein that causes host autolysis, the engineered strain BL21 (DE3::ara) exhibited higher biomass, cell survival rate and foreign protein expression level than that of BL21 (DE3). In addition, these engineered strains had been successfully applied to improve the production of membrane proteins, including E. coli cytosine transporter protein (CodB), the E. coli membrane protein insertase/foldase (YidC), and the E. coli F-ATPase subunit b (Ecb). The engineered strains constructed in this paper provided more host choices for the production of recombinant proteins.

scholarly journals Regulating the T7 RNA Polymerase Expression in E. coli BL21 (DE3) to Provide More Host Options for Recombinant Protein Production

2021 ◽  
Author(s):  
Ying-Shuang Xu ◽  
Fei Du ◽  
Zi-Jia Li ◽  
Yu-Zhou Wang ◽  
Zi-Xu Zhang ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Recombinant Protein ◽  
Rna Polymerase ◽  
Recombinant Proteins ◽  
Protein Production ◽  
T7 Rna Polymerase ◽  
Foreign Protein ◽  
Atpase Subunit ◽  
E Coli ◽  
Prokaryotic Expression System

Abstract Escherichia coli is the most widely used bacterium in prokaryotic expression system for the production of recombinant proteins. In BL21 (DE3), the gene encoding the T7 RNA polymerase (T7 RNAP) is under control of the strong lacUV5 promoter (PLacUV5), which produces more T7 RNAP than wild-type lac promoter (PLacWT) to promote the production of recombinant proteins. However, there is a resource allocated limitation between cell growth and protein production when producing autolytic proteins or membrane proteins. T7 RNAP is the key factor to solve this problem. Hence, we replaced respectively PLacUV5 with other inducible promoters: arabinose promoter (ParaBAD), rhamnose promoter (PrhaBAD), tetracycline promoter (Ptet) to optimize the production of recombinant protein by regulating the transcription level of T7 RNAP. Compared with BL21 (DE3), the constructed engineering strains had higher sensitivity to inducers, among which rhamnose and tetracycline promoters had the lowest leakage ability. In the glucose dehydrogenase (GDH) production, the engineered strains BL21 (DE3::tet) exhibited great biomass, cell survival rate and foreign protein expression level. In addition, these engineered strains had been successfully applied to the production of other membrane proteins, including E. coli cytosine transporter protein (CodB), the E. coli membrane protein insertase/foldase (YidC), and E. coli F-ATPase subunit b (Ecb). The engineering strains constructed in this paper provided more host choices for the production of recombinant proteins.

CRISPR/Cas9 mediated T7 RNA polymerase gene knock-in in E. coli BW25113 makes T7 expression system work efficiently

2021 ◽  
Author(s):  
Changchuan Ye ◽  
Xi Chen ◽  
Mengjie Yang ◽  
Xiangfang Zeng ◽  
Shiyan Qiao
Keyword(s):  
Rna Polymerase ◽  
Mutant Strain ◽  
Aminolevulinic Acid ◽  
Expression System ◽  
T7 Rna Polymerase ◽  
Bacterial Strains ◽  
T7 Promoter ◽  
E Coli ◽  
T7 Expression System ◽  
High Level

Abstract T7 Expression System is a common method of ensuring tight control and high-level induced expression. However, this system can only work in some bacterial strains in which the T7 RNA Polymerase gene resides in the chromosome. In this study, we successfully introduced a chromosomal copy of the T7 RNA Polymerase gene under control of the lacUV5 promoter into Escherichia coli BW25113. The T7 Expression System worked efficiently in this mutant strain named BW25113-T7. We demonstrated that this mutant strain could satisfactorily produce 5-Aminolevulinic Acid via C5 pathway. A final study was designed to enhance the controllability of T7 Expression System in this mutant strain by constructing a T7 Promoter Variants Library. These efforts advanced E. coli BW25113-T7 to be a practical host for future metabolic engineering efforts.

scholarly journals Impact of the Expression System on Recombinant Protein Production in Escherichia coli BL21

2021 ◽  
Vol 12 ◽  
Author(s):  
Gema Lozano Terol ◽  
Julia Gallego-Jara ◽  
Rosa Alba Sola Martínez ◽  
Adrián Martínez Vivancos ◽  
Manuel Cánovas Díaz ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Proteins ◽  
Copy Number ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Recombinant Protein Expression ◽  
Expression System ◽  
E Coli

Recombinant protein production for medical, academic, or industrial applications is essential for our current life. Recombinant proteins are obtained mainly through microbial fermentation, with Escherichia coli being the host most used. In spite of that, some problems are associated with the production of recombinant proteins in E. coli, such as the formation of inclusion bodies, the metabolic burden, or the inefficient translocation/transport system of expressed proteins. Optimizing transcription of heterologous genes is essential to avoid these drawbacks and develop competitive biotechnological processes. Here, expression of YFP reporter protein is evaluated under the control of four promoters of different strength (PT7lac, Ptrc, Ptac, and PBAD) and two different replication origins (high copy number pMB1′ and low copy number p15A). In addition, the study has been carried out with the E. coli BL21 wt and the ackA mutant strain growing in a rich medium with glucose or glycerol as carbon sources. Results showed that metabolic burden associated with transcription and translation of foreign genes involves a decrease in recombinant protein expression. It is necessary to find a balance between plasmid copy number and promoter strength to maximize soluble recombinant protein expression. The results obtained represent an important advance on the most suitable expression system to improve both the quantity and quality of recombinant proteins in bioproduction engineering.

scholarly journals CRISPR/Cas9 mediated T7 RNA polymerase gene knock-in in E. coli BW25113 makes T7 expression system work efficiently

2021 ◽  
Vol 15 (1) ◽  
Author(s):  
Changchuan Ye ◽  
Xi Chen ◽  
Mengjie Yang ◽  
Xiangfang Zeng ◽  
Shiyan Qiao
Keyword(s):  
Rna Polymerase ◽  
Mutant Strain ◽  
Aminolevulinic Acid ◽  
Expression System ◽  
T7 Rna Polymerase ◽  
Bacterial Strains ◽  
T7 Promoter ◽  
E Coli ◽  
T7 Expression System ◽  
High Level

AbstractT7 Expression System is a common method of ensuring tight control and high-level induced expression. However, this system can only work in some bacterial strains in which the T7 RNA Polymerase gene resides in the chromosome. In this study, we successfully introduced a chromosomal copy of the T7 RNA Polymerase gene under control of the lacUV5 promoter into Escherichia coli BW25113. The T7 Expression System worked efficiently in this mutant strain named BW25113-T7. We demonstrated that this mutant strain could satisfactorily produce 5-Aminolevulinic Acid via C5 pathway. A final study was designed to enhance the controllability of T7 Expression System in this mutant strain by constructing a T7 Promoter Variants Library. These efforts advanced E. coli BW25113-T7 to be a practical host for future metabolic engineering efforts.

Expression of Foreign Proteins in Escherichia coli

2021 ◽  
Author(s):  
Ali Iftikhar
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Recombinant Proteins ◽  
Expression System ◽  
Maximum Yield ◽  
Protein Yield ◽  
Foreign Protein ◽  
Low Protein ◽  
Prokaryotic Expression System ◽  
Foreign Proteins

Abstract BackgroundOptimization of conditions for the recombinant production of proteins in a prokaryotic expression system is essential as the recombinant proteins impose a metabolic burden on cell's growth leading to low protein yield and low protein expression resulting from cell death.Main textThe concentration of media components is optimized to accommodate for depleted nutrients due to foreign protein expression. The temperature is optimized to reduce proteolytic degradation and accumulation of protein as inclusion bodies in Escherichia coli. The concentration of inducer and time of induction for high protein yield is also optimized. These optimization conditions depend on the promoter under which the gene of interest is present and the characteristics of the target protein.ConclusionIn the past few years, many optimization conditions for the production of recombinant proteins in Escherichia coli have been studied. These conditions depend mainly upon the promoter used to produce protein and the type of protein produced. Optimizing the expression parameters of protein produced in Escherichia coli ensures maximum yield of the desired protein.

scholarly journals Recent Developments of Recombinant Protein Expression for Therapeutic Purposes

2021 ◽  
Vol 3 (4) ◽  
Author(s):  
Anam Amir
Keyword(s):  
Recombinant Protein ◽  
Recombinant Proteins ◽  
Expression System ◽  
Cell Penetrating Peptides ◽  
Ovary Cell ◽  
Expression Systems ◽  
E Coli ◽  
Mammalian Expression ◽  
Mammalian Expression System ◽  
High Level

In the most recent seven to eight years, the therapeutic recombinant proteins have rapidly expanded in the biotechnology domain due to its wide variety of needs. There has been significant development in the mammalian expression system for fine purification and increased level of expressed recombinant proteins [1,2]. Many drugs like tetracycline have been demonstrated on the Chinese Hamster Ovary cell line for promising multi control strategies and effective cytotoxicity. Mammalian expression system improves the proper glycosylation of recombinant proteins which are very helpful to increase solubility of product [3-6].             Meanwhile on the prokaryotic expression system, E. coli has proven to be an easier to handle, friendly and economical strain [2]. Recently these expression systems are using to work on antibody fragment productions and their proper folding with co-expression of chaperones [7]. Moreover E. coli has been used for the production of cancer cell penetrating peptides which promises the targeted delivery of drugs to specific effector cells only.  Yeast systems are also being used for the antibody fragments production and the high level production of insulin. Interestingly cell free expression systems are also participating in this game and that would be very fascinating to see in the coming years about cell extract medium for production of high level recombinant protein [8, 9]. Purification and optimization of recombinant protein has always been a challenging situation for scientists and they paid more attention to increase the overall yield of the product. Many affinity chromatography techniques has been introduced for efficient purification of protein of interest [10]. Despite these research and developments in methodologies to produce and purify the recombinant therapeutic protein, scientists still face the hurdles and challenges with all expression systems. Rationally E. coli produces inclusion bodies and many mammalian cell types do not show the same results with the same recombinant protein. [11]. So there is a requirement for adding the appropriate features to the expression systems focused to better improvising recovery, production and purification of recombinant protein. Copyright(c) The Author

scholarly journals Multiple Microfermentor Battery: a Versatile Tool for Use with Automated Parallel Cultures of Microorganisms Producing Recombinant Proteins and for Optimization of Cultivation Protocols

2006 ◽  
Vol 72 (8) ◽  
pp. 5225-5231 ◽  
Author(s):  
Emmanuel Frachon ◽  
Vincent Bondet ◽  
Hélène Munier-Lehmann ◽  
Jacques Bellalou
Keyword(s):  
Recombinant Protein ◽  
Recombinant Proteins ◽  
Protein Production ◽  
Batch Cultures ◽  
E Coli ◽  
Working Volume ◽  
Versatile Tool ◽  
Labview Program ◽  
Medium Formulation ◽  
Conventional Medium

ABSTRACT A multiple microfermentor battery was designed for high-throughput recombinant protein production in Escherichia coli. This novel system comprises eight aerated glass reactors with a working volume of 80 ml and a moving external optical sensor for measuring optical densities at 600 nm (OD600) ranging from 0.05 to 100 online. Each reactor can be fitted with miniature probes to monitor temperature, dissolved oxygen (DO), and pH. Independent temperature regulation for each vessel is obtained with heating/cooling Peltier devices. Data from pH, DO, and turbidity sensors are collected on a FieldPoint (National Instruments) I/O interface and are processed and recorded by a LabVIEW program on a personal computer, which enables feedback control of the culture parameters. A high-density medium formulation was designed, which enabled us to grow E. coli to OD600 up to 100 in batch cultures with oxygen-enriched aeration. Accordingly, the biomass and the amount of recombinant protein produced in a 70-ml culture were at least equivalent to the biomass and the amount of recombinant protein obtained in a Fernbach flask with 1 liter of conventional medium. Thus, the microfermentor battery appears to be well suited for automated parallel cultures and process optimization, such as that needed for structural genomics projects.

Rapid E. coli-based cloning and expression system for production of recombinant proteins

2014 ◽  
Vol 185 ◽  
pp. S70
Author(s):  
Boguslaw Lupa ◽  
Krzysztof Stawujak ◽  
Igor Rozanski ◽  
Justyna Stec-Niemczyk
Keyword(s):  
Recombinant Proteins ◽  
Expression System ◽  
Cloning And Expression ◽  
E Coli

scholarly journals Recombinant protein production provoked accumulation of ATP, fructose-1,6-bisphosphate and pyruvate in E. coli K12 strain TG1

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Jan Weber ◽  
Zhaopeng Li ◽  
Ursula Rinas
Keyword(s):  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Expression System ◽  
Limiting Factors ◽  
Expression Vectors ◽  
Metabolic Enzyme ◽  
E Coli ◽  
Metabolic Burden ◽  
Fructose 1,6 Bisphosphate

Abstract Background Recently it was shown that production of recombinant proteins in E. coli BL21(DE3) using pET based expression vectors leads to metabolic stress comparable to a carbon overfeeding response. Opposite to original expectations generation of energy as well as catabolic provision of precursor metabolites were excluded as limiting factors for growth and protein production. On the contrary, accumulation of ATP and precursor metabolites revealed their ample formation but insufficient withdrawal as a result of protein production mediated constraints in anabolic pathways. Thus, not limitation but excess of energy and precursor metabolites were identified as being connected to the protein production associated metabolic burden. Results Here we show that the protein production associated accumulation of energy and catabolic precursor metabolites is not unique to E. coli BL21(DE3) but also occurs in E. coli K12. Most notably, it was demonstrated that the IPTG-induced production of hFGF-2 using a tac-promoter based expression vector in the E. coli K12 strain TG1 was leading to persistent accumulation of key regulatory molecules such as ATP, fructose-1,6-bisphosphate and pyruvate. Conclusions Excessive energy generation, respectively, accumulation of ATP during recombinant protein production is not unique to the BL21(DE3)/T7 promoter based expression system but also observed in the E. coli K12 strain TG1 using another promoter/vector combination. These findings confirm that energy is not a limiting factor for recombinant protein production. Moreover, the data also show that an accelerated glycolytic pathway flux aggravates the protein production associated “metabolic burden”. Under conditions of compromised anabolic capacities cells are not able to reorganize their metabolic enzyme repertoire as required for reduced carbon processing.

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