scholarly journals A freeze-thaw method for disintegration of Escherichia coli cells producing T7 lysozyme used in pBAD expression systems.

2007 ◽  
Vol 54 (3) ◽  
pp. 671-672 ◽  
Author(s):  
Marta Wanarska ◽  
Piotr Hildebrandt ◽  
Józef Kur
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Protein Production ◽  
Protein Gene ◽  
Expression Systems ◽  
E Coli ◽  
Escherichia Coli Cells ◽  
Deinococcus Geothermalis ◽  
Lac Promoter ◽  
Cell Disintegration

The pLysN plasmid containing the T7 lysozyme gene under control of the lac promoter was constructed to facilitate cell disintegration after expression of recombinant proteins in arabinose-induced expression systems. The usefulness of this plasmid was tested in Escherichia coli TOP10 and E. coli LMG194 cells carrying pBADMHADgeSSB plasmid containing Deinococcus geothermalis SSB protein gene under control of the araBAD promoter. The results showed that low-level expression of T7 lysozyme did not interfere with the target SSB protein production, and that the freezing-thawing treatment was sufficient for disruption of the E. coli cells producing low amounts of T7 lysozyme.

scholarly journals Overexpression of Trigger Factor Prevents Aggregation of Recombinant Proteins in Escherichia coli

2000 ◽  
Vol 66 (3) ◽  
pp. 884-889 ◽  
Author(s):  
Kazuyo Nishihara ◽  
Masaaki Kanemori ◽  
Hideki Yanagi ◽  
Takashi Yura
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Protein Production ◽  
Trigger Factor ◽  
Human Lysozyme ◽  
E Coli ◽  
Constructed Plasmids ◽  
Expression Plasmids

ABSTRACT To examine the effects of overexpression of trigger factor (TF) on recombinant proteins produced in Escherichia coli, we constructed plasmids that permitted controlled expression of TF alone or together with the GroEL-GroES chaperones. The following three proteins that are prone to aggregation were tested as targets: mouse endostatin, human oxygen-regulated protein ORP150, and human lysozyme. The results revealed that TF overexpression had marked effects on the production of these proteins in soluble forms, presumably through facilitating correct folding. Whereas overexpression of TF alone was sufficient to prevent aggregation of endostatin, overexpression of TF together with GroEL-GroES was more effective for ORP150 and lysozyme, suggesting that TF and GroEL-GroES play synergistic roles in vivo. Although coexpression of the DnaK-DnaJ-GrpE chaperones was also effective for endostatin and ORP150, coexpression of TF and GroEL-GroES was more effective for lysozyme. These results attest to the usefulness of the present expression plasmids for improving protein production inE. coli.

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 Escherichia coli σ70 promoters allow expression rate control at the cellular level in genome-integrated expression systems

2020 ◽  
Author(s):  
Artur Schuller ◽  
Monika Cserjan-Puschmann ◽  
Christopher Tauer ◽  
Johanna Jarmer ◽  
Martin Wagenknecht ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Expression System ◽  
Cellular Level ◽  
Lac Repressor ◽  
Expression Systems ◽  
E Coli ◽  
Basal Expression

Abstract Background The genome-integrated T7 expression system offers significant advantages, in terms of productivity and product quality, even when expressing the gene of interest (GOI) from a single copy of. Compared to plasmid-based expression systems, this system does not incur a plasmid-mediated metabolic load, and it does not vary the dosage of the GOI during the production process. However, long-term production with T7 expression system leads to a rapidly growing non-producing population, because the T7 RNA polymerase (RNAP) is prone to mutations. The present study aimed to investigate whether two σ 70 promoters, which were recognized by the Escherichia coli host RNAP, might be suitable in genome-integrated expression systems. We applied a promoter engineering strategy that allowed control of expressing the model protein, GFP, by introducing lac operators ( lacO ) into the constitutive T5 and A1 promoter sequences. Results We showed that, in genome-integrated E. coli expression systems that used σ 70 promoters, the number of lacO sites must be well balanced. Promoters containing three and two lacO sites exhibited low basal expression, but resulted in a complete stop in recombinant protein production in partially induced cultures. In contrast, expression systems regulated by a single lacO site and the lac repressor element, lacI Q , on the same chromosome caused very low basal expression, were highly efficient in recombinant protein production, and enables fine-tuning of gene expression levels on a cellular level. Conclusions Based on our results, we hypothesized that this phenomenon was associated with the autoregulation of the lac repressor protein, LacI. We reasoned that the affinity of LacI for the lacO sites of the GOI must be lower than the affinity of LacI to the lacO sites of the endogenous lac operon; otherwise, LacI autoregulation could not take place, and the lack of LacI autoregulation would lead to a disturbance in lac repressor-mediated regulation of transcription. By exploiting the mechanism of LacI autoregulation, we created a novel E. coli expression system for use in recombinant protein production, synthetic biology, and metabolic engineering applications.

scholarly journals Escherichia coli σ70 promoters allow expression rate control at the cellular level in genome-integrated expression systems

2019 ◽  
Author(s):  
Artur Schuller ◽  
Monika Cserjan-Puschmann ◽  
Christopher Tauer ◽  
Johanna Jarmer ◽  
Martin Wagenknecht ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Expression System ◽  
Lac Repressor ◽  
Expression Systems ◽  
E Coli ◽  
Basal Expression ◽  
T7 Expression System

Abstract Background The genome-integrated T7 expression system offers significant advantages, in terms of productivity and product quality, even when expressing the gene of interest (GOI) from a single copy of. Compared to plasmid-based expression systems, this system does not incur a plasmid-mediated metabolic load, and it does not vary the dosage of the GOI during the production process. However, long-term production with T7 expression system leads to a rapidly growing non-producing population, because the T7 RNA polymerase (RNAP) is prone to mutations. The present study aimed to investigate whether two σ 70 promoters, which were recognized by the Escherichia coli host RNAP, might be suitable in genome-integrated expression systems. We applied a promoter engineering strategy that allowed control of expressing the model protein, GFP, by introducing lac operators ( lacO ) into the constitutive T5 and A1 promoter sequences.Results We showed that, in genome-integrated E. coli expression systems that used σ 70 promoters, the number of lacO sites must be well balanced. Promoters containing three and two lacO sites exhibited low basal expression, but resulted in a complete stop in recombinant protein production in partially induced cultures. In contrast, expression systems regulated by a single lacO site and the lac repressor element, lacI Q , on the same chromosome caused very low basal expression, were highly efficient in recombinant protein production, and enables fine-tuning of gene expression levels on a cellular level.Conclusions Based on our results, we hypothesized that this phenomenon was associated with the autoregulation of the lac repressor protein, LacI. We reasoned that the affinity of LacI for the lacO sites of the GOI must be lower than the affinity of LacI to the lacO sites of the endogenous lac operon; otherwise, LacI autoregulation could not take place, and the lack of LacI autoregulation would lead to a disturbance in lac repressor-mediated regulation of transcription. By exploiting the mechanism of LacI autoregulation, we created a novel E. coli expression system for use in recombinant protein production, synthetic biology, and metabolic engineering applications.

scholarly journals Posttranslational Targeting of a Recombinant Protein Promotes Its Efficient Secretion into the Escherichia coli Periplasm

2019 ◽  
Vol 85 (13) ◽  
Author(s):  
A. Jimmy Ytterberg ◽  
Roman A. Zubarev ◽  
Thomas Baumgarten
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Recombinant Proteins ◽  
Protein Production ◽  
Antibody Fragment ◽  
Secretory Proteins ◽  
Content Type ◽  
Biomass Formation ◽  
E Coli ◽  
Sec Translocon

ABSTRACT Many recombinant proteins that are produced in Escherichia coli have to be targeted to the periplasm to be functional. N-terminal signal peptides can be used to direct recombinant proteins to the membrane-embedded Sec translocon, a multiprotein complex that translocates proteins across the membrane into the periplasm. We have recently shown that the cotranslational targeting of the single-chain variable antibody fragment BL1 saturates the capacity of the Sec translocon leading to impaired translocation of secretory proteins and protein misfolding/aggregation in the cytoplasm. In turn, protein production yields and biomass formation were low. Here, we study the consequences of targeting BL1 posttranslationally to the Sec translocon. Notably, the posttranslational targeting of BL1 does not saturate the Sec translocon capacity, and both biomass formation and protein production yields are increased. Analyzing the proteome of cells producing the posttranslationally targeted BL1 indicates that the decreased synthesis of endogenous secretory and membrane proteins prevents a saturation of the Sec translocon capacity. Furthermore, in these cells, highly abundant chaperones and proteases can clear misfolded/aggregated proteins from the cytoplasm, thereby improving the fitness of these cells. Thus, the posttranslational targeting of BL1 enables its efficient production in the periplasm due to a favorable adaptation of the E. coli proteome. We envisage that our observations can be used to engineer E. coli for the improved production of recombinant secretory proteins. IMPORTANCE The bacterium Escherichia coli is widely used to produce recombinant proteins. To fold properly, many recombinant proteins have to be targeted to the E. coli periplasm, but so far the impact of the targeting pathway of a recombinant protein to the periplasm has not been extensively investigated. Here, we show that the targeting pathway of a recombinant antibody fragment has a tremendous impact on cell physiology, ultimately affecting protein production yields in the periplasm and biomass formation. This indicates that studying the targeting and secretion of proteins into the periplasm could be used to design strategies to improve recombinant protein production yields.

scholarly journals Random and combinatorial mutagenesis for improved total production of secretory target protein in Escherichia coli

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
David Gonzalez-Perez ◽  
James Ratcliffe ◽  
Shu Khan Tan ◽  
Mary Chen May Wong ◽  
Yi Pei Yee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Secretion ◽  
Protein Production ◽  
Secretory Protein ◽  
Target Protein ◽  
Carrier Protein ◽  
Signal Peptides ◽  
Carrier Proteins ◽  
E Coli ◽  
Combinatorial Mutagenesis

AbstractSignal peptides and secretory carrier proteins are commonly used to secrete heterologous recombinant protein in Gram-negative bacteria. The Escherichia coli osmotically-inducible protein Y (OsmY) is a carrier protein that secretes a target protein extracellularly, and we have previously applied it in the Bacterial Extracellular Protein Secretion System (BENNY) to accelerate directed evolution. In this study, we reported the first application of random and combinatorial mutagenesis on a carrier protein to enhance total secretory target protein production. After one round of random mutagenesis followed by combining the mutations found, OsmY(M3) (L6P, V43A, S154R, V191E) was identified as the best carrier protein. OsmY(M3) produced 3.1 ± 0.3 fold and 2.9 ± 0.8 fold more secretory Tfu0937 β-glucosidase than its wildtype counterpart in E. coli strains BL21(DE3) and C41(DE3), respectively. OsmY(M3) also produced more secretory Tfu0937 at different cultivation temperatures (37 °C, 30 °C and 25 °C) compared to the wildtype. Subcellular fractionation of the expressed protein confirmed the essential role of OsmY in protein secretion. Up to 80.8 ± 12.2% of total soluble protein was secreted after 15 h of cultivation. When fused to a red fluorescent protein or a lipase from Bacillus subtillis, OsmY(M3) also produced more secretory protein compared to the wildtype. In this study, OsmY(M3) variant improved the extracellular production of three proteins originating from diverse organisms and with diverse properties, clearly demonstrating its wide-ranging applications. The use of random and combinatorial mutagenesis on the carrier protein demonstrated in this work can also be further extended to evolve other signal peptides or carrier proteins for secretory protein production in E. coli.

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.

scholarly journals Induction and control of the autolytic system of Escherichia coli

1982 ◽  
Vol 152 (1) ◽  
pp. 26-34
Author(s):  
M Leduc ◽  
R Kasra ◽  
J van Heijenoort
Keyword(s):  
Escherichia Coli ◽  
Fatty Acid ◽  
Acid Synthesis ◽  
Induction Method ◽  
E Coli ◽  
Escherichia Coli Cells ◽  
Carbonyl Cyanide ◽  
And Control ◽  
First Time ◽  
Induced Autolysis

Various methods of inducing autolysis of Escherichia coli cells were investigated, some being described here for the first time. For the autolysis of growing cells only induction methods interfering with the biosynthesis of peptidoglycan were taken into consideration, whereas with harvested cells autolysis was induced by rapid osmotic or EDTA shock treatments. The highest rates of autolysis were observed after induction by moenomycin, EDTA, or cephaloridine. The different autolyses examined shared certain common properties. In particular, regardless of the induction method used, more or less extensive peptidoglycan degradation was observed, and 10(-2) M Mg2+ efficiently inhibited the autolytic process. However, for other properties a distinction was made between methods used for growing cells and those used for harvested cells. Autolysis of growing cells required RNA, protein, and fatty acid synthesis. No such requirements were observed with shock-induced autolysis performed with harvested cells. Thus, the effects of Mg2+, rifampicin, chloramphenicol, and cerulenin clearly suggest that distinct factors are involved in the control of the autolytic system of E. Coli. Uncoupling agents such as sodium azide, 2,4-dinitrophenol, and carbonyl-cyanide-m-chlorophenyl hydrazone used at their usual inhibiting concentration had no effect on the cephaloridine or shock-induced autolysis.

High-Fidelity Translation of Recombinant Human Hemoglobin in Escherichia coli

1998 ◽  
Vol 64 (5) ◽  
pp. 1589-1593 ◽  
Author(s):  
Michael J. Weickert ◽  
Izydor Apostol
Keyword(s):  
Escherichia Coli ◽  
Error Rates ◽  
High Fidelity ◽  
Human Hemoglobin ◽  
Heterologous Proteins ◽  
Expression Systems ◽  
High Level Expression ◽  
E Coli ◽  
Translation Error ◽  
High Level

ABSTRACT Coexpression of di-α-globin and β-globin in Escherichia coli in the presence of exogenous heme yielded high levels of soluble, functional recombinant human hemoglobin (rHb1.1). High-level expression of rHb1.1 provides a good model for measuring mistranslation in heterologous proteins. rHb1.1 does not contain isoleucine; therefore, any isoleucine present could be attributed to mistranslation, most likely mistranslation of one or more of the 200 codons that differ from an isoleucine codon by 1 bp. Sensitive amino acid analysis of highly purified rHb1.1 typically revealed ≤0.2 mol of isoleucine per mol of hemoglobin. This corresponds to a translation error rate of ≤0.001, which is not different from typical translation error rates found for E. coli proteins. Two different expression systems that resulted in accumulation of globin proteins to levels equivalent to ∼20% of the level of E. colisoluble proteins also resulted in equivalent translational fidelity.

Influence of Curli Expression by Escherichia coli O157:H7 on the Cell's Overall Hydrophobicity, Charge, and Ability To Attach to Lettuce

2007 ◽  
Vol 70 (6) ◽  
pp. 1339-1345 ◽  
Author(s):  
RENEE R. BOYER ◽  
SUSAN S. SUMNER ◽  
ROBERT C. WILLIAMS ◽  
MERLE D. PIERSON ◽  
DAVID L. POPHAM ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Surface Charge ◽  
Extracellular Proteins ◽  
Escherichia Coli O157 ◽  
Fruit And Vegetable ◽  
E Coli ◽  
Iceberg Lettuce ◽  
Escherichia Coli Cells ◽  
Abiotic Surfaces ◽  
Plant Surfaces

Curli fibers are produced by some Escherichia coli cells in response to environmental stimuli. These extracellular proteins enhance the cell's ability to form biofilms on various abiotic surfaces. E. coli O157:H7 cells readily attach to a variety of fruit and vegetable surfaces. It is not known whether the expression of curli influences the cell's ability to attach to produce surfaces. In this study, the effect of curli expression on the cell's overall hydrophobicity, charge, and ability to attach to cut and whole iceberg lettuce surfaces was examined. All strains, regardless of curli expression, attached preferentially to the cut edges of lettuce (P < 0.05). The curli-producing cells of E. coli O157:H7 strain E0018 attached in significantly greater numbers to both cut and whole lettuce pieces than did the non–curli-producing E0018 cells (P < 0.05); however, no significant attachment differences were observed between the curli-producing and non–curli-producing cells of E. coli O157:H7 strains 43894 and 43895. All curli-producing E. coli O157:H7 strains were significantly more hydrophobic (P < 0.01); however, no association between the cells' hydrophobic characteristics and lettuce attachment was observed. Overall surface charge of the cells did not differ among strains or curli phenotypes. Results indicate that overall hydrophobicity and cell charge in E. coli O157:H7 strains do not influence attachment to iceberg lettuce surfaces. The presence of curli may not have any influence on attachment of E. coli O157:H7 cells to produce items. Additional factors may influence the attachment of E. coli O157:H7 to plant surfaces and should be further examined.

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