scholarly journals Cloning, expression and purification of the recombinant FliC from Salmonella enteritidis

2016 ◽  
Vol 19 (4) ◽  
pp. 62-69
Author(s):  
Chau Thi Bao Tran ◽  
Anh Viet Nguyen ◽  
Hieu Van Tran
Keyword(s):  
Escherichia Coli ◽  
Salmonella Enteritidis ◽  
Recombinant Vaccines ◽  
The Novel ◽  
Expression And Purification ◽  
Material Source ◽  
Flic Gene ◽  
Recombinant Vector ◽  
Sds Page ◽  
Adjuvant Property

FliC protein from Salmonella enteritidis is currently interested due to its immunologic adjuvant property for the novel generation of recombinant vaccines. To produce a source for further researches on the immune effects of FliC, we generated an Escherichia coli based on recombinant vector called pET-fliC which is ligated from fliC gene with NdeI and XhoI double digested pET vectors. The results of expression of recombinant FliC, which was induced by IPTG, were confirmed by SDS-PAGE and Western blot probed with anti-6xHis tag. With the purity above 95 %, this recombinant FliC can be used as a material source for next studies on evaluating the adjuvant property of FliC.

scholarly journals CONSTRUCTION, EXPRESSION AND PURIFICATION OF RECOMBINANT PRE-MATURE PEPTIDE OF PLANTARICIN F FROM Lactobacillus plantarum S34 IN Escherichia coli

2015 ◽  
Vol 16 (1) ◽  
pp. 31
Author(s):  
Kusdianawati Kusdianawati ◽  
Apon Zaenal Mustopa ◽  
Suharsono Suharsono ◽  
Bugi Ratno Budiarto ◽  
Fatimah Fatimah ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Lactobacillus Plantarum ◽  
Proteolytic Enzymes ◽  
Leader Peptide ◽  
Human Consumption ◽  
Mature Peptide ◽  
Expression And Purification ◽  
Food Preservative ◽  
E Coli ◽  
Sds Page

Plantaricin is one of bacteriocins that have the potential to be used as food preservative. Plantaricin is safe for human consumption because it can be easily degraded by proteolytic enzymes. The objective of this study was to express and purify recombinant pre-mature peptide of plantaricin F from <em>Lactobacillus plantarum</em> S34 in <em>Escherichia coli</em>. Plantaricin gene-specific primer was used to obtain pln F structural gene amplicon from L. <em>plantarum</em> S34. This amplicon was cloned in pET32a vector and expressed in E. coli BL21 (DE3) pLysS. Pre-mature plantaricin F peptide was expressed as Histagged-fusion protein and separated by Co2+-chelating affinity chromatography. L. <em>plantarum</em> S34-derived pre-mature plantaricin F peptide fused with thioredoxin-(His)6tag had successfully been expressed in E. <em>coli</em> BL21 (DE3) pLysS using pET32a as an expression vector. The fused recombinant pln F as pre-mature state expressed had a molecular mass of +24 kDa, meanwhile the fused recombinant that contained only the leader peptide of pln F appeared as +20 kDa based on SDS-PAGE separations. The optimal production of fused recombinant pln F as soluble fraction was obtained when culture condition was added with 0.5 mM of IPTG and incubated at 22°C for 5 hours (OD~1). Furthermore, the expression of fused recombinant pln F as its pre-mature peptide pointed out that the pln F’s leader peptide could be proteolytically cleaved by a system in heterologous cells. Overall, heterologous pln F production as pre-mature peptide fused with thioredoxin-(His)6tag had been well established. From this research, we expect plantaricin F can be expressed and purified in E. coli.

scholarly journals CONSTRUCTING A NOVEL PLASMID TO INCREASE PURITY EFFECTIVENESS FOR RECOMBINANT PROTEINS EXPRESSED IN ESCHERICHIA COLI CELLS

2021 ◽  
Vol 44 (02) ◽  
Author(s):  
THI-HUYEN TRAN ◽  
HOANG-ANH PHAN THI ◽  
MINH-NAM NGUYEN ◽  
DINH-TRUONG NGUYEN ◽  
LIN-WOO KANG
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
The Novel ◽  
Food Technology ◽  
Purification Process ◽  
Plasmid Construction ◽  
His Tag ◽  
Escherichia Coli Cells ◽  
Sds Page ◽  
Histidine Tag

Nowadays, requirement of supply for recombinant proteins has increased in several fields such as food technology, medical pharmacy, clinical diagnose or environment treatment. The recombinant proteins have become the commercialized products and of that yielded with increasing in a large number per year. Besides, supposing that on extending of the his-tag of the pET111a plasmid may be facilitate for removing his-tag and more effective in protein purification. In this study, nine nucleotides (GCGGCGGCG) coding three alanine residues were added to positions followed hexa-histidine tag (his-tag) on a pET11a plasmid construction. The SDS-PAGE result of each recombinant protein contained the long-modified tag after purification process almost only exhibited single band on gel. Based on alike observed consequences for three recombinant proteins already refined the purity effectiveness reach to upper 98% in the total of existing proteins inside the solution. Hence, the novel pET11a plasmid construction could become an effective plasmid for the aim of harvesting high-purified recombinant proteins.

scholarly journals CONSTRUCTION, EXPRESSION AND PURIFICATION OF RECOMBINANT PRE-MATURE PEPTIDE OF PLANTARICIN F FROM Lactobacillus plantarum S34 IN Escherichia coli

2015 ◽  
Vol 16 (1) ◽  
pp. 31 ◽  
Author(s):  
Kusdianawati Kusdianawati ◽  
Apon Zaenal Mustopa ◽  
Suharsono Suharsono ◽  
Bugi Ratno Budiarto ◽  
Fatimah Fatimah ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Lactobacillus Plantarum ◽  
Proteolytic Enzymes ◽  
Leader Peptide ◽  
Human Consumption ◽  
Mature Peptide ◽  
Expression And Purification ◽  
Food Preservative ◽  
E Coli ◽  
Sds Page

Plantaricin is one of bacteriocins that have the potential to be used as food preservative. Plantaricin is safe for human consumption because it can be easily degraded by proteolytic enzymes. The objective of this study was to express and purify recombinant pre-mature peptide of plantaricin F from <em>Lactobacillus plantarum</em> S34 in <em>Escherichia coli</em>. Plantaricin gene-specific primer was used to obtain pln F structural gene amplicon from L. <em>plantarum</em> S34. This amplicon was cloned in pET32a vector and expressed in E. coli BL21 (DE3) pLysS. Pre-mature plantaricin F peptide was expressed as Histagged-fusion protein and separated by Co2+-chelating affinity chromatography. L. <em>plantarum</em> S34-derived pre-mature plantaricin F peptide fused with thioredoxin-(His)6tag had successfully been expressed in E. <em>coli</em> BL21 (DE3) pLysS using pET32a as an expression vector. The fused recombinant pln F as pre-mature state expressed had a molecular mass of +24 kDa, meanwhile the fused recombinant that contained only the leader peptide of pln F appeared as +20 kDa based on SDS-PAGE separations. The optimal production of fused recombinant pln F as soluble fraction was obtained when culture condition was added with 0.5 mM of IPTG and incubated at 22°C for 5 hours (OD~1). Furthermore, the expression of fused recombinant pln F as its pre-mature peptide pointed out that the pln F’s leader peptide could be proteolytically cleaved by a system in heterologous cells. Overall, heterologous pln F production as pre-mature peptide fused with thioredoxin-(His)6tag had been well established. From this research, we expect plantaricin F can be expressed and purified in E. coli.

scholarly journals SUB-CLONING OF GENES ENCODINGCYTHOCHROME P450 MONOOXYGENASE (CYP71AVI) INTOEXPRESSION VECTOR IN ESCHERICHIA COLI

2017 ◽  
Vol 10 (14) ◽  
pp. 69
Author(s):  
Imam Adi Wicaksono ◽  
Tresna Lestari ◽  
Evi Umayah Ulfa ◽  
Catur Riani ◽  
Elfahmi Elfahmi
Keyword(s):  
Escherichia Coli ◽  
Restriction Site ◽  
Restriction Analysis ◽  
Sequencing Analysis ◽  
Gene Encoding ◽  
P450 Monooxygenase ◽  
Recombinant Vector ◽  
Sds Page ◽  
Key Enzyme ◽  
Artemisinin Biosynthesis

Objective: Cytochrome P450 monooxygenase (CYP71AVI) is a key enzyme involved in the artemisinin biosynthesis pathway.In this research, sub-cloning gene encoding CYP71AVI into pETDUET1 vector in Escherichia coli has been done and then the expression products characterized with SDS-PAGE.Methods: Gene construction started with sub-cloning of cyp71avi gene from pJexpress401_cyp into pETDUET1 through restriction site NdeI and XhoI to get pETDUET1_cyp. Overproduction of CYP71AVI at temperature 37 °C has conducted by IPTG induction.Results: Confirmation of the recombinant vector pETDUET1_cyp was done by migration, restriction site and sequencing analysis. The result of pETDUET1_cyp restriction analysis with XhoI restriction enzyme showed one DNA band with experimental size 6585 bp.The CYP71AVI protein has been produced and characterized with SDS-PAGE method. Based on experimental calculation from SDS-PAGE analysis obtained molecular weight of CYP71AVI band was 57.55 kDa.Conclusion: Construction of gene encoding CYP71AVI into pETDUET1 as the co-expression vector in Escherichia colihas been succesfully and confirmed by migration, restriction site and sequencing analysis. The result of overproduction showed protein bands on SDS-PAGE analysis indicated as CYP71AVI. 

scholarly journals Long-Term Experimental Evolution in Escherichia coli. IV. Targets of Selection and the Specificity of Adaptation

Genetics ◽  
1996 ◽  
Vol 143 (1) ◽  
pp. 15-26 ◽  
Author(s):  
Michael Travisano ◽  
Richard E Lenski
Keyword(s):  
Escherichia Coli ◽  
Experimental Evolution ◽  
Common Ancestor ◽  
The Novel ◽  
Physiological Mechanisms ◽  
Outer Membranes ◽  
Limiting Nutrient ◽  
Novel Environments ◽  
Uptake Mechanisms

Abstract This study investigates the physiological manifestation of adaptive evolutionary change in 12 replicate populations of Escherichia coli that were propagated for 2000 generations in a glucose-limited environment. Representative genotypes from each population were assayed for fitness relative to their common ancestor in the experimental glucose environment and in 11 novel single-nutrient environments. After 2000 generations, the 12 derived genotypes had diverged into at least six distinct phenotypic classes. The nutrients were classified into four groups based upon their uptake physiology. All 12 derived genotypes improved in fitness by similar amounts in the glucose environment, and this pattern of parallel fitness gains was also seen in those novel environments where the limiting nutrient shared uptake mechanisms with glucose. Fitness showed little or no consistent improvement, but much greater genetic variation, in novel environments where the limiting nutrient differed from glucose in its uptake mechanisms. This pattern of fitness variation in the novel nutrient environments suggests that the independently derived genotypes adapted to the glucose environment by similar, but not identical, changes in the physiological mechanisms for moving glucose across both the inner and outer membranes.

Expression and Purification of Bioactive High-Purity Recombinant Mouse SPP1 in Escherichia coli

2014 ◽  
Vol 173 (2) ◽  
pp. 421-432 ◽  
Author(s):  
Yunsheng Yuan ◽  
Xiyuan Zhang ◽  
Shunyan Weng ◽  
Wen Guan ◽  
Di Xiang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Purity ◽  
Expression And Purification ◽  
Recombinant Mouse

Screening of Commercial and Pecan Shell–Extracted Liquid Smoke Agents as Natural Antimicrobials against Foodborne Pathogens

2012 ◽  
Vol 75 (6) ◽  
pp. 1148-1152 ◽  
Author(s):  
ELLEN J. VAN LOO ◽  
D. BABU ◽  
PHILIP G. CRANDALL ◽  
STEVEN C. RICKE
Keyword(s):  
Escherichia Coli ◽  
Foodborne Pathogens ◽  
Salmonella Enteritidis ◽  
Antioxidant Properties ◽  
Inhibitory Effects ◽  
Natural Antimicrobials ◽  
E Coli ◽  
Liquid Smoke ◽  
Water Based ◽  
Smoke Sample

Liquid smoke extracts have traditionally been used as flavoring agents, are known to possess antioxidant properties, and serve as natural alternatives to conventional antimicrobials. The antimicrobial efficacies of commercial liquid smoke samples may vary depending on their source and composition and the methods used to extract and concentrate the smoke. We investigated the MICs of eight commercial liquid smoke samples against Salmonella Enteritidis, Staphylococcus aureus, and Escherichia coli. The commercial liquid smoke samples purchased were supplied by the manufacturer as water-based or concentrated extracts of smoke from different wood sources. The MICs of the commercial smokes to inhibit the growth of foodborne pathogens ranged from 0.5 to 6.0% for E. coli, 0.5 to 8.0% for Salmonella, and 0.38 to 6% for S. aureus. The MIC for each liquid smoke sample was similar in its effect on both E. coli and Salmonella. Solvent-extracted antimicrobials prepared using pecan shells displayed significant differences between their inhibitory concentrations depending on the type of solvent used for extraction. The results indicated that the liquid smoke samples tested in this study could serve as effective natural antimicrobials and that their inhibitory effects depended more on the solvents used for extraction than the wood source.

scholarly journals BACTERIAL AGGLUTINATION BY A LECTIN FROM THE LEAVES OF THE MEDICINAL PLANT, PIMENTA DIOICA (L.) MERR

2018 ◽  
Vol 10 (4) ◽  
pp. 35 ◽  
Author(s):  
Surya P H ◽  
Elyas K K ◽  
Deepti Madayi
Keyword(s):  
Escherichia Coli ◽  
Medicinal Plant ◽  
Biochemical Characterization ◽  
Exchange Chromatography ◽  
Bacterial Typing ◽  
Gram Negative ◽  
Sds Page ◽  
Bacterial Filters ◽  
Pimenta Dioica ◽  
Bacterial Agglutination

Objective: The current investigation involves the purification, characterization of the lectin from the leaves of Pimenta dioica (L.) Merr. (Myrtaceae) a medicinal plant, and its application in bacterial typing.Methods: A lectin was purified from the leaves by cation exchange chromatography. SDS PAGE revealed the molecular weight of the purified lectin. Biochemical characterization was carried out by performing various tests. Hemagglutination inhibition was conducted to detect the sugar specificity. Additionally, bacterial agglutination was performed to predict whether the purified lectin was able to agglutinate the bacterial strains.Results: SDS PAGE analysis revealed the lectin to be a tetramer in the range of 43-66 kDa. The purified lectin agglutinated human, avian, and mouse erythrocytes, and was inhibited by 125 mmol of mannose and xylose. The lectin was stable at 0-60 ° C for 30 min and was unaffected by either 2-Mercaptoethanol (2-ME) or Dithiothreitol (DTT) (50-250µM). A pH of 6.0–8.0 was found optimum for its activity and was nearly independent of metal ions. The purified lectin contained about 20% carbohydrate as estimated by Anthrone method. Purified lectin agglutinated the Gram-negative Escherichia coli and Proteus vulgaris.Conclusion: The isolated lectin was found to possess significant hemagglutinating activity. Due to its ability to agglutinate Gram negative bacteria such as Escherichia coli and Proteus vulgaris, it could be used for bacterial typing and for the design of bacterial filters.

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