scholarly journals Expression comparison between two genes encoding CSF3 recombinant proteins having different codon composition at N-terminal in Escherichia coli

2021 ◽  
Vol 948 (1) ◽  
pp. 012081
Author(s):  
K S Dewi ◽  
F D Wahyuni ◽  
S Salsabila ◽  
Aminah ◽  
N D Yanthi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Recombinant Proteins ◽  
Recombinant Plasmid ◽  
Synonymous Codon ◽  
Expression System ◽  
Negative Control ◽  
Translation Initiation Region ◽  
E Coli ◽  
Genes Encoding

Abstract Colony-stimulating factor 3 (CSF3) is a glycoprotein with many therapeutic applications. In the Escherichia coli expression system, mRNA folding and stability near the translation initiation region (TIR) are known to influence protein expression significantly. We have successfully constructed the recombinant plasmid carrying genes encoding CSF3.1 and CSF3.2, which have different synonymous codon usage at N-terminal. In this study, we compared both expressions of CSF3.1 and CSF3.2 recombinant proteins in E. coli host. Recombinant plasmid pJ414-CSF3.1 and pJ414-CSF3.2 were transformed individually into E. coli NiCo21(DE3) competent cells by a heat-shock method, then spread on solid Lysogeny Broth (LB) medium containing ampicillin. Eight transformant colonies were selected and then expressed in 2xYT medium with the addition of IPTG inducer. Expression analysis was carried out using 15% SDS-PAGE gel. No significantly different band was observed in CSF3.1 protein expression compared to the negative control. In contrast, CSF3.2 protein can be expressed with a good amount at its expected size of 18 kDa. This result was strengthened by bioinformatics analysis which demonstrated the more open TIR of CSF3.2 than that of CSF3.1 Our study highlighted that AU-rich mRNA at the N-terminal is essential for efficient recognition of the ribosome binding site.

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 of Recombinant Fusion Protein of Newcastle Disease Virus from Escherichia coli Plasmid Clone C-2a by In-vitro Cell-free Protein Expression System

2020 ◽  
Vol 20 ◽  
pp. 04004
Author(s):  
Ahmad Pandu Satria Wiratama ◽  
Aris Haryanto
Keyword(s):  
Protein Expression ◽  
Newcastle Disease ◽  
Recombinant Plasmid ◽  
Expression System ◽  
Free Protein ◽  
F Protein ◽  
E Coli ◽  
Protein Expression System ◽  
Sds Page

Newcastle Disease Virus (NDV) is an infectious disease that infect many kinds of wild and domesticated birds. Infection of NDV become a massive problem for poultry industry around the world especially in Indonesia. Vaccination is an effort to prevent the infection of NDV in poultry. NDV vaccine that used in Indonesia is a conventional life vaccine from LaSota and B1 strains. These type of vaccine is 21%-23% genetically distinct with the virus that spread in the environment. The antibody protection provided by the vaccine is not effective. Therefore, vaccination with new local NDV strain is needed to prevent the NDV infection in Indonesia. The previously study research reported that the local isolate of NDV from Kulon Progo, Indonesia has been isolated. Fusion (F) protein encoding gene that has been inserted into pBT7-N-His expression p lasmid which isolated from clone C-2a of E. coli, then it was expressed by the Cell-free protein expression system. The aim of this study was to confirm whether clone C-2a of E.coli carrying a recombinant plasmid pBT7-N-His-Fusion NDV and to express a recombinant F protein of NDV in-vitro from expression plasmid by cell-free protein expression system. This work started by detection of recombinant plasmid pBT7-N-His-Fusion NDV by DNA plasmid extraction followed by agarose gel electrophoresis. The recombinant F protein was in-vitro expressed by cell-free protein expression kit. The expressed F protein of NDV then was visualized by SDS-PAGE and Westernblott to analyse the expression of NDV recombinant F protein. It confirmed that clone C-2a of E. coli contained plasmid pBT7-N-His (4.001 bp) inserted by recombinant F protein of NDV gene (642 bp). The visualisation of expressed recombinant F protein by SDS-PAGE and Westernblott showed the NDV recombinant F protein was a specific protein fragment with molecular weight of 25,6 kDa..

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 Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Masuzu Kikuchi ◽  
Keiichi Kojima ◽  
Shin Nakao ◽  
Susumu Yoshizawa ◽  
Shiho Kawanishi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Proton Pump ◽  
Expression System ◽  
Spectroscopic Characterization ◽  
Functional Expression ◽  
Proton Pumping ◽  
E Coli ◽  
Oxyrrhis Marina ◽  
Domains Of Life

AbstractMicrobial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pKa = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins.

Viability and survival capability of quinolone-resistant uropathogenic Escherichia coli

2010 ◽  
Vol 5 (6) ◽  
pp. 827-830
Author(s):  
Georgi Slavchev ◽  
Nadya Markova
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Expression System ◽  
Antibiotic Sensitivity ◽  
Uropathogenic Escherichia Coli ◽  
Resistant Bacteria ◽  
E Coli ◽  
Serum Bactericidal Assay ◽  
Tract Infections ◽  
Macrophage Killing

AbstractUropathogenic strains of E. coli isolated from urine of patients with urinary tract infections were tested for antibiotic sensitivity using bio-Merieux kits and ATB-UR 5 expression system. The virulence of strains was evaluated by serum bactericidal assay, macrophage “killing” and bacterial adhesive tests. Survival capability of strains was assessed under starvation in saline. The results showed that quinolone-resistant uropathogenic strains of E. coli exhibit significantly reduced adhesive potential but relatively high resistance to serum and macrophage bactericidity. In contrast to laboratory strains, the quinolone-resistant uropathogenic clinical isolate demonstrated increased viability during starvation in saline. Our study suggests that quinolone-resistant uropathogenic strains are highly adaptable clones of E. coli, which can exhibit compensatory viability potential under unfavorable conditions. The clinical occurrence of such phenotypes is likely to contribute to the survival, persistence and spread strategy of resistant bacteria.

scholarly journals Functional Studies of [FeFe] Hydrogenase Maturation in an Escherichia coli Biosynthetic System

2006 ◽  
Vol 188 (6) ◽  
pp. 2163-2172 ◽  
Author(s):  
Paul W. King ◽  
Matthew C. Posewitz ◽  
Maria L. Ghirardi ◽  
Michael Seibert
Keyword(s):  
Escherichia Coli ◽  
Catalytic Domain ◽  
Expression System ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
E Coli ◽  
Functional Studies ◽  
Hydrogenase Maturation ◽  
Iron Sulfur ◽  
And Function

ABSTRACT Maturation of [FeFe] hydrogenases requires the biosynthesis and insertion of the catalytic iron-sulfur cluster, the H cluster. Two radical S-adenosylmethionine (SAM) proteins proposed to function in H cluster biosynthesis, HydEF and HydG, were recently identified in the hydEF-1 mutant of the green alga Chlamydomonas reinhardtii (M. C. Posewitz, P. W. King, S. L. Smolinski, L. Zhang, M. Seibert, and M. L. Ghirardi, J. Biol. Chem. 279:25711-25720, 2004). Previous efforts to study [FeFe] hydrogenase maturation in Escherichia coli by coexpression of C. reinhardtii HydEF and HydG and the HydA1 [FeFe] hydrogenase were hindered by instability of the hydEF and hydG expression clones. A more stable [FeFe] hydrogenase expression system has been achieved in E. coli by cloning and coexpression of hydE, hydF, and hydG from the bacterium Clostridium acetobutylicum. Coexpression of the C. acetobutylicum maturation proteins with various algal and bacterial [FeFe] hydrogenases in E. coli resulted in purified enzymes with specific activities that were similar to those of the enzymes purified from native sources. In the case of structurally complex [FeFe] hydrogenases, maturation of the catalytic sites could occur in the absence of an accessory iron-sulfur cluster domain. Initial investigations of the structure and function of the maturation proteins HydE, HydF, and HydG showed that the highly conserved radical-SAM domains of both HydE and HydG and the GTPase domain of HydF were essential for achieving biosynthesis of active [FeFe] hydrogenases. Together, these results demonstrate that the catalytic domain and a functionally complete set of Hyd maturation proteins are fundamental to achieving biosynthesis of catalytic [FeFe] hydrogenases.

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 Evaluation of antipyretic activity of hydroalcoholic extract of Corchorus depressus Linn. in Escherichia coli–induced pyretic rabbits

2018 ◽  
Vol 16 ◽  
pp. 205873921879295
Author(s):  
Saeed Ahmad ◽  
Muhammad Akram ◽  
Syed Muhammad Ali Shah ◽  
Sabira Sultana
Keyword(s):  
Escherichia Coli ◽  
Positive Control ◽  
Negative Control ◽  
The Body ◽  
Hydroalcoholic Extract ◽  
Antipyretic Effect ◽  
E Coli ◽  
Antipyretic Activity ◽  
Experimental Treatments

This study was conducted to investigate the antipyretic effect of the hydroalcoholic extract of Corchorus depressus Linn. against Escherichia coli ( E. coli)-induced pyrexia in rabbits. Hydroalcohalic extracts of C. depressus were given orally at 25, 50, and 100 mg/kg for antipyretic affect in E. coli-induced fever in rabbits. The animals were divided into five groups of five each. Among these five groups, three received various doses of experimental treatments, whereas the fourth one served as positive control and received paracetamol. The fifth group of animals served as negative control and received no treatment. The body temperature of the rabbits was measured rectally over a period of 5 h. C. depressus exhibited better effects at dose rate of 25, 50, and 100 mg/kg. The hydroalcoholic extract of C. depressus has significant antipyretic effect. These results lend support to the popular use of C. depressus in traditional medicine as a remedy for pyrexia and suggest that the characterization of the principles for such activity deserves further investigation.

scholarly journals Ferric uptake regulator (Fur) reversibly binds a [2Fe-2S] cluster to sense intracellular iron homeostasis in Escherichia coli

2020 ◽  
Vol 295 (46) ◽  
pp. 15454-15463 ◽  
Author(s):  
Chelsey R. Fontenot ◽  
Homyra Tasnim ◽  
Kathryn A. Valdes ◽  
Codrina V. Popescu ◽  
Huangen Ding
Keyword(s):  
Escherichia Coli ◽  
Iron Content ◽  
Iron Homeostasis ◽  
Ferric Uptake Regulator ◽  
Intracellular Iron ◽  
Free Iron ◽  
E Coli ◽  
Genes Encoding ◽  
Fur Protein ◽  
Mutant Cells

The ferric uptake regulator (Fur) is a global transcription factor that regulates intracellular iron homeostasis in bacteria. The current hypothesis states that when the intracellular “free” iron concentration is elevated, Fur binds ferrous iron, and the iron-bound Fur represses the genes encoding for iron uptake systems and stimulates the genes encoding for iron storage proteins. However, the “iron-bound” Fur has never been isolated from any bacteria. Here we report that the Escherichia coli Fur has a bright red color when expressed in E. coli mutant cells containing an elevated intracellular free iron content because of deletion of the iron–sulfur cluster assembly proteins IscA and SufA. The acid-labile iron and sulfide content analyses in conjunction with the EPR and Mössbauer spectroscopy measurements and the site-directed mutagenesis studies show that the red Fur protein binds a [2Fe-2S] cluster via conserved cysteine residues. The occupancy of the [2Fe-2S] cluster in Fur protein is ∼31% in the E. coli iscA/sufA mutant cells and is decreased to ∼4% in WT E. coli cells. Depletion of the intracellular free iron content using the membrane-permeable iron chelator 2,2´-dipyridyl effectively removes the [2Fe-2S] cluster from Fur in E. coli cells, suggesting that Fur senses the intracellular free iron content via reversible binding of a [2Fe-2S] cluster. The binding of the [2Fe-2S] cluster in Fur appears to be highly conserved, because the Fur homolog from Hemophilus influenzae expressed in E. coli cells also reversibly binds a [2Fe-2S] cluster to sense intracellular iron homeostasis.

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