scholarly journals Construction of an AI-2 quorum sensing induced heterologous protein expression system in Escherichia coli

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e12497
Author(s):  
Fei Shang ◽  
Hui Wang ◽  
Dan Zhang ◽  
Wenhui Wang ◽  
Jiangliu Yu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Quorum Sensing ◽  
Protein Expression ◽  
Recombinant Protein Expression ◽  
Expression System ◽  
Heterologous Protein Expression ◽  
Promoter Regions ◽  
T7 Promoter ◽  
Iptg Induction ◽  
Pet Expression System

Background The pET expression system based on T7 promoter which is induced by isopropyl-β-D-1-thiogalactopyranoside (IPTG) is by far the most commonly used system for production of heterogeneous proteins in Escherichia coli. However, this system was limited by obvious drawbacks including the host toxicity and metabolic burden imposed by the presence of IPTG. Methods In this study, we incorporated the autoinducer-2 (AI-2) quorum sensing system to realize autoinduction of the pET expression system. The autoinduction expression vector pXWZ1 was constructed by inserting the lsr promoter regions into the pET28a(+) vector. The expression efficiency of the reporter genes gfpuv and lacZ by the pXWZ1 and pET28a(+) vectors were compared. Results The results showed that the expression levels of the both report genes in the cells transformed with pXWZ1 without any addition of exogenous inducer were higher than that transformed with pET28a(+) vectors by the induction of IPTG. Conclusion This new auto-induction system will exclude the limitations of the IPTG induction including toxic to host and increasing formation of inclusion body and will become a more economical and convenient tool for recombinant protein expression.

scholarly journals Escherichia coli Extract-Based Cell-Free Expression System as an Alternative for Difficult-to-Obtain Protein Biosynthesis

2020 ◽  
Vol 21 (3) ◽  
pp. 928 ◽  
Author(s):  
Sviatlana Smolskaya ◽  
Yulia A. Logashina ◽  
Yaroslav A. Andreev
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Dna ◽  
Recombinant Protein Expression ◽  
Expression System ◽  
Reaction Medium ◽  
Cellular Metabolism ◽  
Heterologous Protein Expression

Before utilization in biomedical diagnosis, therapeutic treatment, and biotechnology, the diverse variety of peptides and proteins must be preliminarily purified and thoroughly characterized. The recombinant DNA technology and heterologous protein expression have helped simplify the isolation of targeted polypeptides at high purity and their structure-function examinations. Recombinant protein expression in Escherichia coli, the most-established heterologous host organism, has been widely used to produce proteins of commercial and fundamental research interests. Nonetheless, many peptides/proteins are still difficult to express due to their ability to slow down cell growth or disrupt cellular metabolism. Besides, special modifications are often required for proper folding and activity of targeted proteins. The cell-free (CF) or in vitro recombinant protein synthesis system enables the production of such difficult-to-obtain molecules since it is possible to adjust reaction medium and there is no need to support cellular metabolism and viability. Here, we describe E. coli-based CF systems, the optimization steps done toward the development of highly productive and cost-effective CF methodology, and the modification of an in vitro approach required for difficult-to-obtain protein production.

scholarly journals The Impact of IPTG Induction on Plasmid Stability and Heterologous Protein Expression by Escherichia coli Biofilms

2020 ◽  
Vol 21 (2) ◽  
pp. 576 ◽  
Author(s):  
Luciana Gomes ◽  
Gabriel Monteiro ◽  
Filipe Mergulhão
Keyword(s):  
Escherichia Coli ◽  
Heterologous Protein ◽  
Fluorescent Protein ◽  
Plasmid Stability ◽  
Gene Dosage ◽  
Heterologous Protein Expression ◽  
Chemical Induction ◽  
T7 Promoter ◽  
Iptg Induction ◽  
The Impact

This work assesses the effect of chemical induction with isopropyl β-D-1-thiogalactopyranoside (IPTG) on the expression of enhanced green fluorescent protein (eGFP) by planktonic and biofilm cells of Escherichia coli JM109(DE3) transformed with a plasmid containing a T7 promoter. It was shown that induction negatively affected the growth and viability of planktonic cultures, and eGFP production did not increase. Heterologous protein production was not limited by gene dosage or by transcriptional activity. Results suggest that plasmid maintenance at high copy number imposes a metabolic burden that precludes high level expression of the heterologous protein. In biofilm cells, the inducer avoided the overall decrease in the amount of expressed eGFP, although this was not correlated with the gene dosage. Higher specific production levels were always attained with biofilm cells and it seems that while induction of biofilm cells shifts their metabolism towards the maintenance of heterologous protein concentration, in planktonic cells the cellular resources are directed towards plasmid replication and growth.

scholarly journals A self-inducible heterologous protein expression system in Escherichia coli

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
L. Briand ◽  
G. Marcion ◽  
A. Kriznik ◽  
J. M. Heydel ◽  
Y. Artur ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Heterologous Protein ◽  
Expression System ◽  
Heterologous Protein Expression ◽  
Protein Expression System

scholarly journals Construction of a Low-Temperature Protein Expression System Using a Cold-Adapted Bacterium, Shewanella sp. Strain Ac10, as the Host

2007 ◽  
Vol 73 (15) ◽  
pp. 4849-4856 ◽  
Author(s):  
Ryoma Miyake ◽  
Jun Kawamoto ◽  
Yun-Lin Wei ◽  
Masanari Kitagawa ◽  
Ikunoshin Kato ◽  
...  
Keyword(s):  
Low Temperature ◽  
Protein Expression ◽  
Recombinant Protein Expression ◽  
Expression System ◽  
Maximum Yield ◽  
Broad Host Range ◽  
Psychrophilic Bacterium ◽  
T7 Promoter ◽  
Cold Adapted ◽  
Protein Expression System

ABSTRACT A recombinant protein expression system working at low temperatures is expected to be useful for the production of thermolabile proteins. We constructed a low-temperature expression system using an Antarctic cold-adapted bacterium, Shewanella sp. strain Ac10, as the host. We evaluated the promoters for proteins abundantly produced at 4°C in this bacterium to express foreign proteins. We used 27 promoters and a broad-host-range vector, pJRD215, to produce β-lactamase in Shewanella sp. strain Ac10. The maximum yield was obtained when the promoter for putative alkyl hydroperoxide reductase (AhpC) was used and the recombinant cells were grown to late stationary phase. The yield was 91 mg/liter of culture at 4°C and 139 mg/liter of culture at 18°C. We used this system to produce putative peptidases, PepF, LAP, and PepQ, and a putative glucosidase, BglA, from a psychrophilic bacterium, Desulfotalea psychrophila DSM12343. We obtained 48, 7.1, 28, and 5.4 mg/liter of culture of these proteins, respectively, in a soluble fraction. The amounts of PepF and PepQ produced by this system were greater than those produced by the Escherichia coli T7 promoter system.

scholarly journals Engineered chromosome-based T7 RNA polymerase in Escherichia coli W3110 for orthogonal T7 promoter circuit as a cell factory

2020 ◽  
Author(s):  
Wan-Wen Ting ◽  
Shih-I Tan ◽  
I-Son Ng
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Rna Polymerase ◽  
Green Fluorescence Protein ◽  
T7 Rna Polymerase ◽  
Cell Factory ◽  
Chemical Production ◽  
T7 Promoter ◽  
Iptg Induction ◽  
Shock Proteins

Abstract Background Orthogonal T7 RNA polymerase (T7RNAP) and T7 promoter were powerful tools to mediate the protein expression. Moreover, Escherichia coli W3110 strain possesses more advantages than the B strain due to more heat shock proteins and higher tolerance to chemicals. Therefore, implementation of T7-based system in W3110 strain is a conceivable strategy to develop the cell factory. Results Three novel W3110 strains with chromosome-equipped T7RNAP (i.e W3110:IL5, W3110::L5 and W3110::pI) were engineered to demonstrate the feasibility on protein expression and chemical production. At first, the LacZ and T7RNAP with IPTG induction showed higher expression levels in W3110 derivatives than that in BL21(DE3). The plasmids with and without lacI/lacO repression were used to investigate the protein expression of super-fold green fluorescence protein (sfGFP), Cas9, carbonic anhydrase (CA) and lysine decarboxylase (CadA). All the proteins were expressed higher and enzymatic functions were better in W3110::L5 and W3110::pI. Moreover, the highest cadaverine production, lysine consumption and the yield were obtained in W3110::L5(+) strain with pET28a(+)-CadA which reached 32.2 g/L, 45 g/L and 91.7% at 24 h, while the W3110::pI(-) strain with pSU-T7-CadA achieved 36.9 g/L, 43.8 g/L and 103.4% at 12 h which is unnecessary of inducer. Conclusion Inducer and lacI/lacO regulators on chromosome and plasmid have been investigated in W3110 strains with T7RNAP. The newly engineered W3110::L5 and W3110:pI both possessed similar protein expression compared to commercial BL21(DE3). Furthermore, among all strains, W3110::pI displayed the greatest potential as cell factory in the future.

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 Expression and Characterization of Recombinant β-1,3-Glucanase of Burkholderia cepacia [BiogenCC E76] Expressed in Escherichia coli Expression Systems

2019 ◽  
Vol 15 (1) ◽  
pp. 35
Author(s):  
Tri Puji Priyatno ◽  
Fitriani Winangsih ◽  
Ifa Manzila ◽  
Maria Bintang
Keyword(s):  
Escherichia Coli ◽  
Burkholderia Cepacia ◽  
Specific Activity ◽  
Expression System ◽  
Pathogenic Fungi ◽  
Start Codon ◽  
Fungal Cell ◽  
T7 Promoter ◽  
Iptg Induction ◽  
Escherichia Coli Expression

<p>Burkholderia cepacia (Bcc) BiogenCC E76 isolate is an endophytic bacterium producing cell wall degrading enzyme, glucanase, and antagonistic to fungal pathogens, such as Magnaporte grisea and Colletotrichum gloeosporioides. The<br />glucanase is able to lyse fungal cell walls composed of glucan causing disintegrity of mycelia and fungi fail to infect plants. The purpose of this study was to clone, express, and characterize 48 kDa subunit of β-1,3-glucanase from Bcc isolate BiogenCC E76 using the Escherichia coli expression system. The 1,300 bp of the β-1,3-glucanase gene was constructed using the pET-32b<br />vector in BamHI-HindIII restriction sites to generate the pET-Glu plasmid. The gene was fused with nucleotides sequence encoding Trx-tag, His-tag, and S-tag producing 65 kDa of recombinant β-1,3-glucanase. Gene expression in the construct was controlled by the T7 promoter and Trx-tag start codon through IPTG induction. The recombinant β-1,3-glucanase was then purified and its activities were tested at different pH and temperature conditions. Results showed that E. coli carrying pET-Glu overexpressed a 65 kDa protein in induced culture as a soluble protein that was expressed in periplasm. Purification result of the crude extract of the recombinant protein obtained 27% pure enzymes with a specific activity of 1,207.976 U/mg and purity level of 3.9 fold. This recombinant glucanase demonstrated optimal activity at 40°C and pH 5–7. A deeper study is needed to understand the role of 48 kDa subunit of β-1,3-glucanase has in antagonistic mechanism of Bcc against pathogenic fungi.</p>

scholarly journals The Protocatechuate 3,4-Dioxygenase Solubility (PCDS) Tag Enhances the Expression and Solubility of Heterogenous Proteins in Escherichia coli

2021 ◽  
Vol 12 ◽  
Author(s):  
Lei Zou ◽  
Sha Li ◽  
Nan Li ◽  
Shi-Long Ruan ◽  
Jing Chen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Heterologous Protein ◽  
Fluorescent Protein ◽  
Recombinant Protein Expression ◽  
Yellow Fluorescent Protein ◽  
Fluorescence Emission ◽  
Soluble Expression ◽  
Heterologous Protein Expression ◽  
E Coli

Escherichia coli has been developed as the most common host for recombinant protein expression. Unfortunately, there are still some proteins that are resistant to high levels of heterologous soluble expression in E. coli. Protein and peptide fusion tags are one of the most important methods for increasing target protein expression and seem to influence the expression efficiency and solubility as well. In this study, we identify a short 15-residue enhancing solubility peptide, the PCDS (protocatechuate 3,4-dioxygenase solubility) tag, which enhances heterologous protein expression in E. coli. This PCDS tag is a 45-bp long sequence encoding a peptide tag involved in the soluble expression of protocatechuate 3,4-dioxygenase, encoded by the pcaHG98 genes of Pseudomonas putida NCIMB 9866. The 45-bp sequence was also beneficial for pcaHG98 gene amplification. This tag was shown to be necessary for the heterologous soluble expression of PcaHG98 in E. coli. Purified His6-PcaHG98e04-PCDS exhibited an activity of 205.63±14.23U/mg against protocatechuate as a substrate, and this activity was not affected by a PCDS tag. This PCDS tag has been fused to the mammalian yellow fluorescent protein (YFP) to construct YFP-PCDS without its termination codons and YFPt-PCDS with. The total protein expressions of YFP-PCDS and YFPt-PCDS were significantly amplified up to 1.6-fold and 2-fold, respectively, compared to YFP alone. Accordingly, His6-YFP-PCDS and His6-YFPt-PCDS had 1.6-fold and 3-fold higher soluble protein yields, respectively, than His6-YFP expressed under the same conditions. His6-YFP, His6-YFP-PCDS, and His6-YFPt-PCDS also showed consistent fluorescence emission spectra, with a peak at 530nm over a scanning range from 400 to 700nm. These results indicated that the use of the PCDS tag is an effective way to improve heterologous protein expression in E. coli.

scholarly journals Development of chromosome-based T7 RNA polymerase and orthogonal T7 promoter circuit in Escherichia coli W3110 as a cell factory

2020 ◽  
Vol 7 (1) ◽  
Author(s):  
Wan-Wen Ting ◽  
Shih-I Tan ◽  
I-Son Ng
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Rna Polymerase ◽  
Green Fluorescence Protein ◽  
T7 Rna Polymerase ◽  
Cell Factory ◽  
T7 Promoter ◽  
Iptg Induction ◽  
E Coli ◽  
A Cell

Abstract Background Orthogonal T7 RNA polymerase (T7RNAP) and T7 promoter is a powerful genetic element to mediate protein expression in different cells. Among all, Escherichia coli possess advantages of fast growth rate, easy for culture and comprehensive elements for genetic engineering. As E. coli W3110 owns the benefits of more heat shock proteins and higher tolerance to toxic chemicals, further execution of T7-based system in W3110 as cell factory is a conceivable strategy. Results Three novel W3110 strains, i.e., W3110:IL5, W3110::L5 and W3110::pI, were accomplished by chromosome-equipped T7RNAP. At first, the LacZ and T7RNAP with isopropyl-β-D-thiogalactopyranoside (IPTG) induction showed higher expression levels in W3110 derivatives than that in BL21(DE3). The plasmids with and without lacI/lacO repression were used to investigate the protein expression of super-fold green fluorescence protein (sfGFP), carbonic anhydrase (CA) for carbon dioxide uptake and lysine decarboxylase (CadA) to produce a toxic chemical cadaverine (DAP). All the proteins showed better expression in W3110::L5 and W3110::pI, respectively. As a result, the highest cadaverine production of 36.9 g/L, lysine consumption of 43.8 g/L and up to 100% yield were obtained in W3110::pI(−) with plasmid pSU-T7-CadA constitutively. Conclusion Effect of IPTG and lacI/lacO regulator has been investigated in three chromosome-based T7RNAP E. coli strains. The newly engineered W3110 strains possessed similar protein expression compared to commercial BL21(DE3). Furthermore, W3110::pI displays higher production of sfGFP, CA and CadA, due to it having the highest sensitivity to IPTG, thus it represents the greatest potential as a cell factory.

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