scholarly journals A dual prokaryotic (E. coli) expression system (pdMAX)

PLoS ONE ◽  
2021 ◽  
Vol 16 (10) ◽  
pp. e0258553
Author(s):  
Manabu Murakami ◽  
Agnieszka M. Murakami ◽  
Shirou Itagaki
Keyword(s):  
Prokaryotic Expression ◽  
Gene Expression Analysis ◽  
Fluorescent Protein ◽  
Expression System ◽  
The Novel ◽  
Iptg Induction ◽  
Dna Topoisomerase ◽  
E Coli ◽  
Effective Selection ◽  
Dna Insertion

In this study, we introduced an efficient subcloning and expression system with two inducible prokaryotic expression promoters, arabinose and lac, in a single plasmid in Escherichia coli. The arabinose promoter unit allows for the expression of a FLAG-tagged protein, while the isopropyl-β-D-thiogalactoside (IPTG)-inducible unit allows for the expression of a Myc-tagged protein. An efficient subcloning (DNA insertion) system (iUnit) follows each promoter. The iUnit, based on a toxin that targets DNA topoisomerase of E. coli, allows for effective selection with arabinose or IPTG induction. With the dual promoter plasmid (pdMAX) system, expressed lacZ (β-galactosidase) activity was significantly decreased compared with the original solo expression system. Despite this disadvantage, we believe that the pdMAX system remains useful. A recombinant plasmid (pdMAX/ara/DsRed/IPTG/EGFP; pdMAX/DsRed/EGFP) with DsRed in the arabinose expression unit and EGFP in the IPTG expression unit showed fluorescent protein expression following additional low-temperature incubation. Thus, the novel pdMAX system allowed efficient subcloning of two different genes and can be used to induce and analyze the expression of two distinct genes. The proposed system can be applied to various types of prokaryotic gene expression analysis.

scholarly journals A dual prokaryotic (E. coli) expression system (pdMAX)

2020 ◽  
Author(s):  
Manabu Murakami ◽  
Agnieszka M. Murakami ◽  
Kazuyoshi Hirota ◽  
Shirou Itagaki
Keyword(s):  
Expression System ◽  
Peptide Sequence ◽  
The Novel ◽  
Iptg Induction ◽  
Dna Topoisomerase ◽  
E Coli ◽  
Restriction Sites ◽  
Effective Selection ◽  
Dna Insertion ◽  
Limited Protein

AbstractIn this study, we introduced an efficient subcloning and expression system with two inducible prokaryotic expression promoters, arabinose and lac, in a single plasmid in Escherichia coli. This pdMAX system is a manageable size at 5811 bp. The arabinose promoter unit allows for the expression of a FLAG-tagged protein, while the isopropyl-β-D-thiogalactoside (IPTG)-inducible unit allows for the expression of a Myc-tagged protein. An efficient subcloning (DNA insertion) system (iUnit) follows each promoter. The iUnit, based on a toxin that targets DNA topoisomerase of E. coli, allows for effective selection with arabinose or IPTG induction.Interestingly, the dual induction plasmid system shows limited protein expression. To analyze the expression of inserted genes, we performed an α-complementation assay, in which the α-peptide of the lacZ (β-galactosidase) gene is inserted in XL-10 E. coli cells. E. coli expressing the recombinant plasmid form blue colonies when plated on ampicillin/IPTG- or arabinose/X-gal-containing plates if the α-peptide was correctly inserted in-frame to produce the tagged protein. This assay system assesses whether the α-peptide was inserted at the restriction sites (EcoRV or SmaI), whether the inserted peptide was expressed, and whether the inserted α-peptide sequence was ligated in-frame to produce a tagged protein. Frameshifts result in no α-complementation and white colonies.With the dual promoter plasmid (pdMAX) system, expressed lacZ activity was significantly decreased comparing with the solo expression (pgMAX) system. Despite this disadvantage, we believe the pdMAX system is still useful for the analysis of distinct genes in E. coli, which will enable different types of expression analysis. Overall, the novel pdMAX system allows for efficient subcloning of two different genes. Furthermore, the pdMAX system could be used to induce and analyze the expression of two distinct genes and adopted to various types of prokaryotic gene expression analyses.

scholarly journals A new dual prokaryotic (E. coli) and mammalian expression system (pgMAXs)

2020 ◽  
Author(s):  
Manabu Murakami ◽  
Agnieszka M. Murakami ◽  
Kazuyoshi Hirota ◽  
Shirou Itagaki
Keyword(s):  
Topoisomerase Ii ◽  
Fluorescent Proteins ◽  
Expression System ◽  
Simple Expression ◽  
Amino Acid Sequences ◽  
Expression Plasmid ◽  
Iptg Induction ◽  
E Coli ◽  
Mammalian Expression ◽  
Effective Selection

AbstractWe introduce an efficient subcloning and expression plasmid system with two different modes (prokaryotic for expression in Escherichia coli with lac promoter and mammalian modes with cytomegalovirus promoter). The efficient subcloning (DNA insertion) is based upon a DNA topoisomerase II toxin-originated gene for effective selection with isopropyl-β-D-thiogalactoside (IPTG) induction. The new pgMAXs system is manageable size (4452 bp) and has also various types of protein tags (flag, myc, poly-histidine, Human influenza hemagglutinin, strep, and v5) for expression analysis. With pgMAXs system, various types of fluorescent proteins were subcloned and prtein expressions were confirmed. We also tried to identify epitope amino acid sequences for anti-calcium channel β2 antibody, by constructing epitope-library with DNaseI-partial digestion and subcloning into EcoRV site in pgMAXs. The new pgMAXs plasmid system enables highly efficient subcloning, simple expression in E. coli and that it has a simple deletion step of rare 8-nucleotide rare-cutter blunt-end enzymes for mammalian expression plasmid construction. Taken together, the pgMAXs system simplifies prokaryotic and mammalian gene expression analyses.

scholarly journals Vaccine Efficacy of Bm86 Ortholog ofH. a. anatolicum, rHaa86 Expressed in Prokaryotic Expression System

2009 ◽  
Vol 2009 ◽  
pp. 1-7 ◽  
Author(s):  
P. Azhahianambi ◽  
D. D. Ray ◽  
Pallab Chaudhuri ◽  
Rohita Gupta ◽  
Srikanta Ghosh
Keyword(s):  
Vaccine Efficacy ◽  
Prokaryotic Expression ◽  
Integrated Control ◽  
Expression System ◽  
Fusion Tag ◽  
E Coli ◽  
Adult Females ◽  
Prokaryotic Expression System ◽  
Integrated Tick Management ◽  
Tick Vaccine

The use of tick vaccine in controlling ticks and tick borne diseases has been proved effective in integrated tick management format. For the control ofH. a. anatolicum, Bm86 ortholog ofH. a. anatolicumwas cloned and expressed as fusion protein inE. coliasE. coli-pETHaa86. The molecular weight of the rHaa86 was 97 kDa with a 19 kDa fusion tag of thioredoxin protein. The expressed protein was characterized immunologically and vaccine efficacy was evaluated. After 120 hours of challenge, only 26% tick could successfully fed on immunized animals. Besides significant reduction in feeding percentages, a significant reduction of 49.6 mg;P<.01in the weight of fed females in comparison to the females fed on control animals was recorded. Following oviposition, a significant reduction of 68.1 mg;P<.05in the egg masses of ticks fed on immunized animals in comparison to the ticks fed on control animals was noted. The reduction of number of females, mean weight of eggs, adult females and efficacy of immunogen were 73.8%, 31.3%, 15.8%, and 82.3%, respectively. The results indicated the possibility of development of rHaa86 based vaccine as a component of integrated control of tick species.

scholarly journals Expression of Lon-like Protease Gene from Lactobacillus plantarum IIA-1A5 in Escherichia coli BL21(DE3)

2019 ◽  
Vol 19 (2) ◽  
pp. 149-158
Author(s):  
Olfa Mega ◽  
Cece Sumantri ◽  
Irma Isnafia Arief ◽  
Cahyo Budiman
Keyword(s):  
Molecular Weight ◽  
Lactobacillus Plantarum ◽  
Expression System ◽  
Gc Content ◽  
Whole Genome Sequence ◽  
Protease Gene ◽  
Iptg Induction ◽  
E Coli ◽  
Sds Page ◽  
Index Value

Proteases are one of most important and abundant enzymes produced by the biotechnology industry, for scientific, physiological and industrial application and dominates of the whole enzyme market. Lactobacillus plantarum IIA-1A5 is an Indonesian lactic acid bacteria (LAB) isolated from beef Peranakan Ongole cattle. Preliminary analysis on its whole genome sequence indicated that this strain harbours some genes involved in protein degradation and might be promising to be further applied. This study aims to optimize the gene sequence of a lon-like protease of L. plantarum IIA-1A5 for heterologous expression system. The Lon-like gene expression system is made using genes that have been optimized first in silico.  pET-28a(+), E. coli BL21(DE3), Nde1 and BamH1 were used in this study as a expression vector, a host and retriction enzyme, respectively.  Molecular weight was validated using SDS-PAGE and expasy.org software. The results showed that optimization increased codon adaptation index value (CAI) and GC content to 0.97 and 56.57%, respectively, which were suitable for the E. coli expression system. The Lon-like IIA gene was successfully expressed in the cell cytoplasm by induction of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37 °C.  As many as 88% of Lon-like IIA codons were distributed in the 91-100 quality group. Lon-like IIA was successfully expressed in a host cell induced with 1 mM IPTG at 37oC . IPTG induction was performed at the 3rd hour of incubation with OD600 0.59. In addition, Lon-like IIA molecular weight was detected approximately 43 kDa.

Indipendent and Tandem Expression of a Novel Antimicrobial Peptides Plectasin in Escherichia coli

2011 ◽  
Vol 345 ◽  
pp. 134-138 ◽  
Author(s):  
Li Hui Lv ◽  
Xue Gang Luo ◽  
Meng Ni ◽  
Xiao Lan Jing ◽  
Nan Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Streptococcus Pneumoniae ◽  
Expression System ◽  
High Yield ◽  
Gene Synthesis ◽  
Post Translational Modification ◽  
Iptg Induction ◽  
E Coli ◽  
Conventional Antibiotics

Plectasin, a novel antimicrobial peptide, is isolated from a saprophytic fungus Pseudoplectania nigrella. Plectasin showed potent antibacterial activity in vitro against Gram-positive, especially the Streptococcus pneumoniae and Streptococcus pneumoniae, including strains resistant to conventional antibiotics. In our previous study, plectasin had been expressed at a high yield as a thioredoxin (Trx) – fused protein in Escherichia coli. However, it couldn’t exhibit the antimicrobial activity unless the Trx-tag had been cleaved, which made the producing process be complicated. Concerning that plectasin has no complex post-translational modification and toxicity on E. coli, on the basis of the former works, we further establish the independent and tandem expression system of plectasin in E. coli. In the present study, the coding sequence of plectasin was obtained from pET32a-PLEC with four primers to amplify the independent and tandem plectasin fragments by overlapping PCR-based gene synthesis, and then cloned into pET22b (+) vector. The recombinant protein was expressed successfully in E. coli with IPTG induction. These works might throw light on the production or study of plectasin, and contribute to the development of novel anti-infectious drugs in the future.

Efficient selection of IgG Fc domain-binding peptides fused to fluorescent protein using E. coli expression system and dot-blotting assay

Peptides ◽  
2010 ◽  
Vol 31 (2) ◽  
pp. 202-206 ◽  
Author(s):  
Yu-jin Jeong ◽  
Hyo Jin Kang ◽  
Kwang-Hee Bae ◽  
Min-Gon Kim ◽  
Sang J. Chung
Keyword(s):  
Fluorescent Protein ◽  
Expression System ◽  
E Coli ◽  
Binding Peptides ◽  
Efficient Selection ◽  
Dot Blotting ◽  
Selection Of

scholarly journals Rapid screening of high expressing Escherichia coli colonies using a novel dicistronic-autoinducible system

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Fatemeh Sadat Shariati ◽  
Dariush Norouzian ◽  
Vahideh Valizadeh ◽  
Reza Ahangari Cohan ◽  
Malihe Keramati
Keyword(s):  
Fluorescent Protein ◽  
Limit Of Detection ◽  
Pearson Correlation ◽  
Expression System ◽  
Rapid Screening ◽  
Limit Of Quantification ◽  
Fluorescent Intensity ◽  
E Coli ◽  
Protein Model ◽  
Green Fluorescent

Abstract Background Identification of high-expressing colonies is one of the main concerns in the upstream process of recombinant protein development. The common method to screen high-producing colonies is SDS-PAGE, a laborious and time-consuming process, which is based on a random and qualitative way. The current study describes the design and development of a rapid screening system composed of a dicistronic expression system containing a reporter (enhanced green fluorescent protein, eGFP), protein model (staphylokinase, SAK), and a self-inducible system containing heat shock protein 27 (Hsp27). Results Dicistronic-autoinducible system expressed eGFP and SAK successfully in 5-ml and 1-L culture volumes. High expressing colonies were identified during 6 h via fluorescent signals. In addition, the biological activity of the protein model was confirmed semi-quantitatively and quantitatively through radial caseinolytic and chromogenic methods, respectively. There was a direct correlation between eGFP fluorescent intensity and SAK activity. The correlation and linearity of expression between the two genes were respectively confirmed with Pearson correlation and linear regression. Additionally, the precision, limit of detection (LOD), and limit of quantification (LOQ) were determined. The expression of eGFP and SAK was stable during four freeze–thaw cycles. In addition, the developed protocol showed that the transformants can be inoculated directly to the culture, saving time and reducing the error-prone step of colony picking. Conclusion The developed system is applicable for rapid screening of high-expressing colonies in most research laboratories. This system can be investigated for other recombinant proteins expressed in E. coli with a potential capability for automation and use at larger scales.

scholarly journals Metabolomic analysis of riboswitch containing E. coli recombinant expression system

2016 ◽  
Vol 12 (2) ◽  
pp. 350-361 ◽  
Author(s):  
Howbeer Muhamadali ◽  
Yun Xu ◽  
Rosa Morra ◽  
Drupad K. Trivedi ◽  
Nicholas J. W. Rattray ◽  
...  
Keyword(s):  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Recombinant Expression ◽  
Expression System ◽  
Metabolic Effects ◽  
Metabolomic Analysis ◽  
E Coli ◽  
System P ◽  
Recombinant Expression System ◽  
Green Fluorescent

In this study we have employed metabolomics approaches to understand the metabolic effects of producing enhanced green fluorescent protein (eGFP) as a recombinant protein inEscherichia colicells.

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