Dual-Expression Vectors for Efficient Protein Expression in Both E. coli and Mammalian Cells

2003 ◽  
pp. 19-30
Author(s):  
Rebecca L. Mullinax ◽  
David T. Wong ◽  
Heidi A. Davis ◽  
Kerstein A. Padgett ◽  
Joseph A. Sorge
Keyword(s):  
Protein Expression ◽  
Mammalian Cells ◽  
Expression Vectors ◽  
E Coli ◽  
Dual Expression

Co-Expression of Proteins in E. coli Using Dual Expression Vectors

2003 ◽  
pp. 205-214
Author(s):  
Karen Johnston ◽  
Ronen Marmorstein
Keyword(s):  
Expression Vectors ◽  
E Coli ◽  
Dual Expression

Effect of supplementing Moringa oleifera essential oils on milk quality and fatty acid profile in dairy sheep

2019 ◽  
Author(s):  
N. Hemamalini ◽  
S. Ezhilmathi ◽  
A. Angela Mercy
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Cell Biology ◽  
Genetic Manipulation ◽  
Recombinant Protein Expression ◽  
Coli Strain ◽  
Expression Vectors ◽  
Functional Domain ◽  
Post Translational Modifications ◽  
E Coli

Escherichia coli is the most extensively used organism in recombinant protein production. It has several advantages including a very short life cycle, ease of genetic manipulation and the well-known cell biology etc. which makes E. coli as the perfect host for recombinant protein expression. Despite many advantages, E. coli also have few disadvantages such as coupled transcription and translation and lack of eukaryotic post-translational modifications. These challenges can be overcome by adopting several strategies such as, using different E. coli expression vectors, changing the gene sequence without altering the functional domain, modified E. coli strain usage, changing the culture parameters and co-expression with a molecular chaperone. In this review, we present the level of strategies used to enhance the recombinant protein expression and its stability in E. coli.

scholarly journals A simple and dual expression plasmid system in prokaryotic (E. coli) and mammalian cells

PLoS ONE ◽  
2019 ◽  
Vol 14 (5) ◽  
pp. e0216169 ◽  
Author(s):  
Manabu Murakami ◽  
Takayoshi Ohba ◽  
Agnieszka M. Murakami ◽  
Chong Han ◽  
Kenji Kuwasako ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Expression Plasmid ◽  
E Coli ◽  
Dual Expression Plasmid ◽  
Dual Expression

scholarly journals PRODUCTION AND BIOACTIVITY POTENTIAL OF THREE RECOMBINANT GROWTH HORMONES OF FARMED FISH

2010 ◽  
Vol 5 (1) ◽  
pp. 11 ◽  
Author(s):  
Alimuddin Alimuddin ◽  
Indra Lesmana ◽  
Agus Oman Sudrajat ◽  
Odang Carman ◽  
Irvan Faizal
Keyword(s):  
Growth Hormone ◽  
Body Weight ◽  
Protein Expression ◽  
Common Carp ◽  
Nile Tilapia ◽  
Expression Vector ◽  
Expression Vectors ◽  
Farmed Fish ◽  
Recombinant Growth Hormone ◽  
E Coli

This study was aimed to produce recombinant growth hormone (rGH) from giant grouper (Epinephelus lanceolatus), giant gouramy (Osphronemus gouramy) and common carp (Cyprinus carpio) and compare their bioactivity potential by means of inducing the growth hormone of juvenile Nile tilapia (Oreochromis niloticus) as the model. DNA fragment encoding mature GH protein of giant grouper (El-mGH), giant gouramy (Og-mGH) and common carp (Cc-mGH) was amplified by PCR method. The purified PCR products were ligated to pCold-1 to generate pCold/El-mGH, pCold/OgmGH, and pCold/Cc-mGH protein expression vector, respectively. Each of the expression vectors was transformed into the Escherichia coli BL21. E. coli BL21 was cultured using 2xYT medium and protein production was induced by cold shock at 15±1oC for overnight. The inclusion bodies of E. coli transformants containing protein expression vector were isolated by sonication method, and rGH production was analyzed by SDS-PAGE. Juvenile of Nile tilapia of average body weight of 12.41±3.28 g was intramuscularly injected once a week for 4 weeks with 1 μg inclusion body containing rGH per gram fish body weight. The result showed that rGH in molecular weight of about 25 kDa was obtained. Fish injected with rGH of El-mGH, Cc-mGH and Og-mGH grew 20.94%, 18.09%, and 16.99% faster, respectively, compared with the control. This result indicated that the three rGH produced in E. coli possessed biological activity when tested on Nile tilapia and further research is needed to find its effect on the growth of other aquaculture fish species.

scholarly journals A simple and dual expression plasmid system in prokaryotic (E. coli) and mammalian cells.

2020 ◽  
Vol 93 (0) ◽  
pp. 3-P-331
Author(s):  
Manabu Murakami ◽  
Takayoshi Ohba ◽  
Agnieszka M. Murakami ◽  
Shirou Itagaki
Keyword(s):  
Mammalian Cells ◽  
Expression Plasmid ◽  
E Coli ◽  
Dual Expression Plasmid ◽  
Dual Expression

Use of Uranium-Labeled Antibodies for Electron Microscopic Localization of Various Antigens in Mammalian Cells

1967 ◽  
Vol 25 ◽  
pp. 136-137
Author(s):  
J. P. Petrali ◽  
E. J. Donati ◽  
L. A. Sternberger
Keyword(s):  
Endoplasmic Reticulum ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Specific Antiserum ◽  
Electron Microscopic ◽  
E Coli ◽  
Cytoplasmic Membranes ◽  
Viral Progeny ◽  
Ultrathin Sections ◽  
Electron Microscopic Localization

Specific contrast is conferred to subcellular antigen by applying purified antibodies, exhaustively labeled with uranium under immunospecific protection, to ultrathin sections. Use of Seligman’s principle of bridging osmium to metal via thiocarbohydrazide (TCH) intensifies specific contrast. Ultrathin sections of osmium-fixed materials were stained on the grid by application of 1) thiosemicarbazide (TSC), 2) unlabeled specific antiserum, 3) uranium-labeled anti-antibody and 4) TCH followed by reosmication. Antigens to be localized consisted of vaccinia antigen in infected HeLa cells, lysozyme in monocytes of patients with monocytic or monomyelocytic leukemia, and fibrinogen in the platelets of these leukemic patients. Control sections were stained with non-specific antiserum (E. coli).In the vaccinia-HeLa system, antigen was localized from 1 to 3 hours following infection, and was confined to degrading virus, the inner walls of numerous organelles, and other structures in cytoplasmic foci. Surrounding architecture and cellular mitochondria were unstained. 8 to 14 hours after infection, antigen was localized on the outer walls of the viral progeny, on cytoplasmic membranes, and free in the cytoplasm. Staining of endoplasmic reticulum was intense and focal early, and weak and diffuse late in infection.

Cell-Specific Chemical Delivery Using a Selective Nitroreductase–Nitroaryl Pair

2018 ◽  
Author(s):  
Todd D. Gruber ◽  
Chithra Krishnamurthy ◽  
Jonathan B. Grimm ◽  
Michael R. Tadross ◽  
Laura M. Wysocki ◽  
...  
Keyword(s):  
Small Molecules ◽  
Mammalian Cells ◽  
Target Cells ◽  
Specific Chemical ◽  
E Coli ◽  
Enzyme Substrate ◽  
Cell Specificity ◽  
General Chemical ◽  
Increased Activity ◽  
Enzyme Variant

<p>The utility of<b> </b>small molecules to probe or perturb biological systems is limited by the lack of cell-specificity. ‘Masking’ the activity of small molecules using a general chemical modification and ‘unmasking’ it only within target cells could overcome this limitation. To this end, we have developed a selective enzyme–substrate pair consisting of engineered variants of <i>E. coli</i> nitroreductase (NTR) and a 2‑nitro-<i>N</i>-methylimidazolyl (NM) masking group. To discover and optimize this NTR–NM system, we synthesized a series of fluorogenic substrates containing different nitroaromatic masking groups, confirmed their stability in cells, and identified the best substrate for NTR. We then engineered the enzyme for improved activity in mammalian cells, ultimately yielding an enzyme variant (enhanced NTR, or eNTR) that possesses up to 100-fold increased activity over wild-type NTR. These improved NTR enzymes combined with the optimal NM masking group enable rapid, selective unmasking of dyes, indicators, and drugs to genetically defined populations of cells.</p>

scholarly journals Regulatory Influence of Galanin and GALR1/GALR2 Receptors on Inflamed Uterus Contractility in Pigs

2021 ◽  
Vol 22 (12) ◽  
pp. 6415
Author(s):  
Barbara Jana ◽  
Jarosław Całka ◽  
Bartosz Miciński
Keyword(s):  
Protein Expression ◽  
Domestic Animals ◽  
Receptor Subtypes ◽  
E Coli ◽  
Regulatory Influence ◽  
Pretreatment Period ◽  
Uterine Inflammation ◽  
Myometrial Contractility

Uterine inflammation is a very common and serious pathology in domestic animals, the development and progression of which often result from disturbed myometrial contractility. We investigated the effect of inflammation on the protein expression of galanin (GAL) receptor subtypes (GALR)1 and GALR2 in myometrium and their role in the contractile amplitude and frequency of an inflamed gilt uterus. The gilts of the E. coli and SAL groups received E. coli suspension or saline in their uteri, respectively, and only laparotomy was performed (CON group). Eight days later, the E. coli group developed severe acute endometritis and lowered GALR1 protein expression in the myometrium. Compared to the pretreatment period, GAL (10−7 M) reduced the amplitude and frequency in myometrium and endometrium/myometrium of the CON and SAL groups, the amplitude in both stripes and frequency in endometrium/myometrium of the E. coli group. In this group, myometrial frequency after using GAL increased, and it was higher than in other groups. GALR2 antagonist diminished the decrease in amplitude in myometrium and the frequency in endometrium/myometrium (SAL, E. coli groups) induced by GAL (10−7 M). GALR1/GALR2 antagonist and GAL (10−7 M) reversed the decrease in amplitude and diminished the decrease in frequency in both examined stripes (CON, SAL groups), and diminished the drop in amplitude and abolished the rise in the frequency in the myometrium (E. coli group). In summary, the inflammation reduced GALR1 protein expression in pig myometrium, and GALR1 and GALR2 participated in the contractile regulation of an inflamed uterus.

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