[6] Use of T7 RNA polymerase to direct expression of cloned genes

Studier, F. William; Rosenberg, Alan H.; Dunn, John J.; Dubendorff, John W.

doi:10.1016/0076-6879(90)85008-c

Cited by 6,142 publications

(301 citation statements)

References 24 publications

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“…In addition this vector contains a Kozak consensus sequence (9) for efficient initiation of translation in eukaryotic hosts. The presence of the p10 baculoviral promoter and the flanking lef 2 (ORF 603) and ORF1629 baculoviral recombination sites allow the construction of recombinant baculoviruses and, finally, a T7 polymerase promoter with lac O operator offers high level inducible expression in E.coli harbouring the λ (DE3) prophage (10). …”

Section: Methodsmentioning

confidence: 99%

A versatile ligation-independent cloning method suitable for high-throughput expression screening applications

Berrow¹,

Alderton²,

Sainsbury³

et al. 2007

Nucleic Acids Research

494

444

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This article describes the construction of a set of versatile expression vectors based on the In-Fusion™ cloning enzyme and their use for high-throughput cloning and expression screening. Modifications to commonly used vectors rendering them compatible with In-Fusion™ has produced a ligation-independent cloning system that is (1) insert sequence independent (2) capable of cloning large PCR fragments (3) efficient over a wide (20-fold) insert concentration range and (4) applicable to expression in multiple hosts. The system enables the precise engineering of (His6-) tagged constructs with no undesirable vector or restriction-site-derived amino acids added to the expressed protein. The use of a multiple host-enabled vector allows rapid screening in both E. coli and eukaryotic hosts (HEK293T cells and insect cell hosts, e.g. Sf9 cells). These high-throughput screening activities have prompted the development and validation of automated protocols for transfection of mammalian cells and Ni-NTA protein purification.

show abstract

Section: Methodsmentioning

confidence: 99%

A versatile ligation-independent cloning method suitable for high-throughput expression screening applications

Berrow¹,

Alderton²,

Sainsbury³

et al. 2007

Nucleic Acids Research

494

444

View full text Add to dashboard Cite

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“…The recA gene was generated by PCR using E.coli MG1655 genomic DNA as a template and cloned into the pET-11c vector (38). DNA sequence of the open reading frame was confirmed by dideoxy DNA sequencing (data not shown).…”

Section: Methodsmentioning

confidence: 99%

“…Mutant gyrA genes, gyrA D82G and gyrA S83W , were constructed using the overlap extension PCR technique (43) and cloned into the pET-11c vector (38). DNA sequences of open reading frames were confirmed by dideoxy DNA sequencing (data not shown).…”

Section: Methodsmentioning

confidence: 99%

“…DNA sequences of open reading frames were confirmed by dideoxy DNA sequencing (data not shown). The GyrA D82G and GyrA S83W proteins were overexpressed in E.coli BL21(DE3) (38) and purified by the protocol used to purify the wild-type GyrA protein (41,42). Purified GyrA D82G and GyrA S83W were mixed with the wild-type GyrB to reconstitute GyrA D82G gyrase and GyrA S83W gyrase, respectively.…”

Section: Methodsmentioning

confidence: 99%

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RecA can stimulate the relaxation activity of topoisomerase I: Molecular basis of topoisomerase-mediated genome-wide transcriptional responses in Escherichia coli

Reckinger¹,

Jeong

Khodursky

et al. 2006

Nucleic Acids Research

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The superhelicity of the chromosome, which is controlled by DNA topoisomerases, modulates global gene expression. Investigations of transcriptional responses to the modulation of gyrase function have identified two types of topoisomerase-mediated transcriptional responses: (i) steady-state changes elicited by a mutation in gyrase, such as the D82G mutation in GyrA, and (ii) dynamic changes elicited by the inhibition of gyrase. We hypothesize that the steady-state effects are due to the changes in biochemical properties of gyrase, whereas the dynamic effects are due to an imbalance between supercoiling and relaxation activities, which appears to be influenced by the RecA activity. Herein, we present biochemical evidence for hypothesized mechanisms. GyrA D82G gyrase exhibits a reduced supercoiling activity. The RecA protein can influence the balance between supercoiling and relaxation activities either by interfering with the activity of DNA gyrase or by facilitating the relaxation reaction. RecA has no effect on the supercoiling activity of gyrase but stimulates the relaxation activity of topoisomerase I. This stimulation is specific and requires formation of an active RecA filament. These results suggest that the functional interaction between RecA and topoisomerase I is responsible for RecA-mediated modulation of the relaxation-dependent transcriptional activity of the Escherichia coli chromosome.

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“…Protein expression and purification Growth and induction of E. coli were performed essentially according to the recommendations by Studier et al [11]. E. coli transformant cells were induced with 1 mM isopropyl b-thiogalactoside (IPTG) for 5 h. Then bacterial lysates in 10 mM Tris-Cl, pH 8.0 and 150 mM NaCl were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie brilliant blue.…”

Section: Plasmid Constructsmentioning

confidence: 99%

Recombinant core proteins of Japanese encephalitis virus as activators of the innate immune response

Chen

Fang

Shih³

et al. 2008

Virus Genes

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Nitric oxide (NO) has been shown to suppress Japanese encephalitis virus (JEV) RNA synthesis, viral protein accumulation, and virus release from infected cells. In this article, the potential viral structural proteins as the activators of NO product were studied at the molecular level. First, the genomic region encoding the JEV structural proteins was cloned into a prokaryotic expression vector pET for high-level expression. After purification, these JEV recombinant proteins were added to macrophages to examine the productions of NO and pro-inflammatory mediators. In this study, the recombinant core protein, but not envelope (E), could trigger NO and pro-inflammatory mediators (TNF-alpha, IL-6, and IL-12) productions on macrophages. And their effects were about 85-95% relative to LPS-stimulated macrophages in a dose-dependent manner. Meanwhile, the rCore-2D could up regulate promoters of IL-8 and TNF-alpha via EGFP expression in reporter plasmid (IL-8p-EGFP and TNF-alphap-EGFP)-transfected cells by flow cytometric analysis. These results suggest that JEV core protein could regulate pro-inflammatory mediators and NO production, and may play a crucial role in the innate immunity for the host to restrict the initial stage of JEV infection.

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[6] Use of T7 RNA polymerase to direct expression of cloned genes

Cited by 6,142 publications

References 24 publications

A versatile ligation-independent cloning method suitable for high-throughput expression screening applications

A versatile ligation-independent cloning method suitable for high-throughput expression screening applications

RecA can stimulate the relaxation activity of topoisomerase I: Molecular basis of topoisomerase-mediated genome-wide transcriptional responses in Escherichia coli

Recombinant core proteins of Japanese encephalitis virus as activators of the innate immune response

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