A novel strategy for production of a highly expressed recombinant protein in an active form

Blackwell, John; Horgan, R.

doi:10.1016/0014-5793(91)81372-f

Cited by 202 publications

(127 citation statements)

References 12 publications

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“…The first identification of a gene encoding a CK de novo biosynthetic enzyme was carried out in the gall-forming bacteria Agrobacterium tumefaciens (Akiyoshi et al 1984;Barry et al 1984). This gene, designated Tmr (tumour morphology root), is located in the T-DNA of the tumour-inducing (Ti) plasmid and encodes an IPT that is primarly associated with synthesis of iPRMP from DMAPP and AMP (Blackwell and Horgan 1991), but with the same K m value that also recognises 4-hydroxy-3-methyl-2-(E)-butenyl diphosphate (HMBPP) as the side chain substrate . In the virulence region of the Agrobacterium tumefaciens Ti-plasmid, which is not translocated to the plant cells, another gene encoding the enzyme of transzeatin synthesis (Tzs) is present, utilising HMBPP as a prenyl donor and AMP as the acceptor (Krall et al 2002).…”

Section: Ck Biosynthesismentioning

confidence: 99%

Cytokinins - recent news and views of evolutionally old molecules

Spíchal¹

2012

Functional Plant Biol.

159

169

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Cytokinins (CKs) are evolutionally old and highly conserved low-mass molecules that have been identified in almost all known organisms. In plants, they evolved into an important group of plant hormones controlling many physiological and developmental processes throughout the whole lifespan of the plant. CKs and their functions are, however, not unique to plants. In this review, the strategies and mechanisms of plants – and phylogenetically distinct plant-interacting organisms such as bacteria, fungi, nematodes and insects employing CKs or regulation of CK status in plants – are described and put into their evolutionary context. The major breakthroughs made in the last decade in the fields of CK biosynthesis, degradation and signalling are also summarised.

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Section: Ck Biosynthesismentioning

confidence: 99%

Cytokinins - recent news and views of evolutionally old molecules

Spíchal¹

2012

Functional Plant Biol.

159

169

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“…Bach1 was expressed in Escherichia coli BL21(DE3) cells grown in Luria Bertani medium containing 1 M sorbitol and 250 M betaine to enhance production of native, folded Bach1 protein and to increase the yield of soluble protein (31). The cells were transformed with human, His-tagged Bach1 clone previously described (22).…”

Section: Expression Of Soluble Recombinant Bach1 Proteinmentioning

confidence: 99%

“…Determination of the stoichiometry of binding of Bach1 to each DNA sequence (32) is outside the scope of this report where the focus is on comparing and enlarging the size of the Bach1/MARE-ARE family of genes and providing the first insight to differences in the Bach1 inter- actions among them. Sorbital and betaine, known to improve the solubility of recombinant proteins expressed in E. coli (31), were used in this study to obtain soluble Bach1 protein that could be isolated without urea. All or any of these factors, solubility, absence of urea, and/or a long V5 tag or competing protein may relate to the binding of Bach1 to MARE/ARE DNA in the absence of small Maf proteins.…”

Section: Bach1 Binds Mare/are Dna Sequences Selectively With Sensitivmentioning

confidence: 99%

Bach1 Repression of Ferritin and Thioredoxin Reductase1 Is Heme-sensitive in Cells and in Vitro and Coordinates Expression with Heme Oxygenase1, β-Globin, and NADP(H) Quinone (Oxido) Reductase1

Hintze¹,

Katoh

Igarashi

et al. 2007

Journal of Biological Chemistry

102

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Ferritin gene transcription is regulated by heme as is ferritin mRNA translation, which is mediated by the well studied mRNA⅐IRE/IRP protein complex. The heme-sensitive DNA sequence in ferritin genes is the maf recognition/antioxidant response element present in several other genes that are induced by heme and repressed by Bach1. We now report that chromatin immunoprecipitated with Bach1 antiserum contains ferritin DNA sequences. In addition, overexpression of Bach1 protein in the transfected cells decreased ferritin expression, indicating insufficient endogenous Bach1 for full repression; decreasing Bach1 with antisense RNA increased ferritin expression. Thioredoxin reductase1, a gene that also contains a maf recognition/ antioxidant response element but is less studied, responded similarly to ferritin, as did the positive controls heme oxygenase1 and NADP(H) quinone (oxido) reductase1. Bach1-DNA promoter interactions in cells were confirmed in vitro with soluble, recombinant Bach1 protein and revealed a quantitative range of Bach1/DNA stabilities: ferritin L ϳ ferritin H ϳ ␤-globin, ␤-globin ϳ 2-fold >heme oxygenase1 ‫؍‬ quinone reductase ␤-globin ϳ 4-fold >thioredoxin reductase1. Such results indicate the possibility that modulation of cellular Bach1 concentrations will have variable effects among the genes coordinately regulated by maf recognition/antioxidant response elements in iron/oxygen/antioxidant metabolism.Genes encoding proteins that manage proteins of iron and oxygen traffic and metabolism are at the nexus of chemical reactions that are both critical and dangerous to life. The iron porphyrin complex, heme, has emerged as a key signal for iron and oxygen metabolism genes, including ferritin L (ftl) 3 (1) and ferritin H (fth) (2). Transcriptional regulation of NADP(H) quinone (oxido) reductase (qr) (3), heme oxygenase1 (ho1) (4), and ␤-globin (5) by heme requires the maf recognition/antioxidant response element (MARE/ARE), a conserved regulatory sequence found in the promoter or enhancer, and the heme binding transcriptional repressor Bach1.Both ftl and fth contain heme-responsive canonical MARE/ ARE promoter sequences (1, 2). The role of Bach1 in hemeregulated ferritin transcription is not known. By contrast, the role of IRP1 and IRP2 in heme-regulated ftl and fth mRNA translation is known (6 -9). The translational mechanism uses IRP1 and IRP2 to coordinate fth and ftl mRNA regulation with that of several other mRNAs important in iron and oxygen homeostasis by binding to iron-responsive elements (IRE) in each of the mRNAs. The IRE is a specific three-dimensional loop-helix-loop-helix structure (10) in the noncoding regions of the mRNAs (11-16). Specific interactions between the iron regulatory proteins IRP1 and IRP2 and the different IREs in the mRNAs create a natural, combinatorial array of RNA⅐protein complexes (17). In the case of ferritin, when IRP1 or 2 are bound to the mRNA the ability of eukaryotic initiation factor 4F to recruit translational machinery to the mRNA is compromised and translat...

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“…This results in enhancement of protein stability (19,20). It has been shown with several proteins that some osmolytes, when added to the growth media, assist protein folding resulting in increased protein solubility (21)(22)(23). Preadaptation of E. coli cells to increasingly high concentrations of salt in the media leads to crosstalk between osmolytes and heat-shock proteins and decreases the tendency of soluble proteins to form insoluble aggregates during heat-shock (24).…”

Section: Introductionmentioning

confidence: 99%

Effect of osmotic stress and heat shock in recombinant protein overexpression and crystallization

Oganesyan¹,

Ankoudinova²,

Kim³

et al. 2007

Protein Expression and Purification

106

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Overexpressed recombinant proteins in bacteria often tend to misfold and accumulate as soluble aggregates and/or inclusion bodies. A strategy for improving the level of expression of recombinant proteins in a soluble native form is to increase the cellular concentration of osmolytes or of chaperones. This can be accomplished by growing the bacterial cells in the presence of high salt, sorbitol, and betaine as well as exposing the cells to a heat shock step. Our results suggest that by growing the cells under varied conditions one may be able to express targets as soluble proteins (from previously insoluble targets) and to improve the chances of their crystallization.

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A novel strategy for production of a highly expressed recombinant protein in an active form

Cited by 202 publications

References 12 publications

Cytokinins - recent news and views of evolutionally old molecules

Cytokinins - recent news and views of evolutionally old molecules

Bach1 Repression of Ferritin and Thioredoxin Reductase1 Is Heme-sensitive in Cells and in Vitro and Coordinates Expression with Heme Oxygenase1, β-Globin, and NADP(H) Quinone (Oxido) Reductase1

Effect of osmotic stress and heat shock in recombinant protein overexpression and crystallization

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