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

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.

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Intracellular degradation of recombinant proteins in relation to their location in Escherichia coli cells

Journal of Biotechnology ◽

10.1016/0168-1656(87)90072-1 ◽

1987 ◽

Vol 5 (1) ◽

pp. 77-86 ◽

Cited By ~ 31

Author(s):

Kazuaki Kitano ◽

Shigeru Fujimoto ◽

Masafumi Nakao ◽

Takuya Watanabe ◽

Yoshio Nakao

Keyword(s):

Escherichia Coli ◽

Recombinant Proteins ◽

Intracellular Degradation ◽

Escherichia Coli Cells

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

Science and Technology Development Journal ◽

10.32508/stdj.v19i4.652 ◽

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.

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Purification, and activity of Human rhinovirus 3C protease fused with N-terminal GST-tag and C-terminal His-tag (GST-HRV3C-His) expressed in Escherichia coli

Tạp chí Khoa học ◽

10.54607/hcmue.js.16.3.2468(2019) ◽

2019 ◽

Vol 16 (3) ◽

pp. 162

Author(s):

Le Duong Vuong ◽

Le Thi Tuong Vy ◽

Phan Thi Phuong Trang ◽

Nguyen Duc Hoang

Keyword(s):

Escherichia Coli ◽

Specific Activity ◽

Human Rhinovirus ◽

Purification Process ◽

Recognition Sequence ◽

Fusion Tag ◽

E Coli ◽

Specific Recognition ◽

3C Protease ◽

His Tag

The Human rhinovirus 3C protease (HRV3C) is one of the most effective enzymes for removing fusion tag in purification process. This protease is often produced as fusion form GST-HRV3C but there is no study about the fusion form: GST-HRV3C-His. In this study, researchers conducted the purification GST-HRV3C-His expressed in E. coli, checked the activity and investigated its application. GST-HRV3C-His could be purified using His-tag column with 86.6% purity and GST column with 96.87%. The specific activity of GST-HRV3C-His was demonstrated to be about 4500 U/mg and its application in the purification of another proteins carrying HRV3C-specific recognition sequence, LEVLFQ¯GP based on His-tag or GST-tag was also proved in this study.

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

Acta Biochimica Polonica ◽

10.18388/abp.2007_3241 ◽

2007 ◽

Vol 54 (3) ◽

pp. 671-672 ◽

Cited By ~ 2

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.

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Comparative Evaluation of a Novel Phage Protein Ligand Assay and Immunomagnetic Separation Method To Isolate the Seven Top Serogroups of Escherichia coli (O157, O26, O103, O145, O111, O45, and O121) in Foods at Risk

Journal of Food Protection ◽

10.4315/0362-028x.jfp-16-479 ◽

2017 ◽

Vol 80 (12) ◽

pp. 1973-1979

Author(s):

M. Bouvier ◽

S. D. Miszczycha ◽

X. Guillarme ◽

C. Tadla ◽

L. Collinet ◽

...

Keyword(s):

Escherichia Coli ◽

Recombinant Proteins ◽

Comparative Evaluation ◽

Pathogenic Bacteria ◽

Detection Method ◽

Immunomagnetic Separation ◽

Escherichia Coli O157 ◽

The Novel ◽

Concentration Method ◽

Phage Protein

ABSTRACT The presence of Shiga toxin–producing Escherichia coli (STEC) in food is a major concern for food safety authorities and industries. Methods for detecting these pathogenic bacteria are crucial. Enrichment of foods for STEC identification has been optimized, but selective concentration of bacteria before isolation still needs to be improved. In the present study, we tested the performance of the VIDAS ESPT detection method against that of the immunomagnetic separation (IMS) method. A preenrichment inoculation was performed to provide a realistic scenario of the contamination that occurs in foods, and the methods were then compared. Results obtained were then confirmed in naturally contaminated foods. Preenrichment inoculation assays revealed that the novel concentration method using phage recombinant proteins or the selective capture of the target top seven STEC serogroups is as specific and sensitive as IMS. Subsequent evaluation of naturally contaminated samples confirmed that the novel concentration method and IMS are equivalent in performance under the conditions tested.

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Growth phase-regulated expression of bolA and morphology of stationary-phase Escherichia coli cells are controlled by the novel sigma factor sigma S.

Journal of Bacteriology ◽

10.1128/jb.173.14.4474-4481.1991 ◽

1991 ◽

Vol 173 (14) ◽

pp. 4474-4481 ◽

Cited By ~ 214

Author(s):

R Lange ◽

R Hengge-Aronis

Keyword(s):

Escherichia Coli ◽

Stationary Phase ◽

Growth Phase ◽

Sigma Factor ◽

The Novel ◽

Escherichia Coli Cells ◽

Regulated Expression

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Measurement of binding strength between prey proteins interacting with Toxoplasma gondii SAG1 and SAG2 using isothermal titration calorimetry (ITC)

Acta Parasitologica ◽

10.1515/ap-2018-0012 ◽

2018 ◽

Vol 63 (1) ◽

pp. 106-113 ◽

Cited By ~ 3

Author(s):

Meng-Yee Lai ◽

Yee-Ling Lau

Keyword(s):

Recombinant Proteins ◽

Fusion Proteins ◽

Titration Calorimetry ◽

Yeast Two Hybrid ◽

Binding Strength ◽

His Tag ◽

Sds Page ◽

Human Proteins ◽

Binding Curve ◽

Two Hybrid

Abstract Following the outcome from a previously performed yeast two-hybrid experiment, the binding strength between T. gondii SAG1 and SAG2 and their respective prey proteins were further confirmed in this study. The sag1, sag2 and their prey genes were amplified and cloned into a pGEMT vector. To express the recombinant proteins, the fragments were then subcloned into a pRSETA vector and transformed into E. coli BL21 (DE3) cells. The recombinant proteins were expressed optimally at 37°C and 1mM of IPTG. The 6X His-tag fusion proteins were purified, dialyzed and concentrated. To confirm the expressed proteins, the recombinant proteins were analysed by SDS-PAGE and Western blot. As expected, the size of SAG1, SAG2, HLY and HZF protein were 32, 23, 28 and 37 kDa, respectively. The purified proteins were loaded onto a MicroCal Auto-iTC200 calorimeter from MicroCal™ to quantify binding strength. ITC results indicated there was a typical binding curve for interactions between SAG1 and HLY protein. However, there was an atypical binding curve obtained for interactions between SAG2 and HZF protein. By observing the data obtained from the ITC assay, both of the human proteins (HLY and HZF) were demonstrated to bind to their respective SAG1 and SAG2 proteins.

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