Parameters for effective <i>in vitro</i> production of zinc finger nucleic acid‐binding proteins

Belak, Zachery R.; Nair, Manoj S.; Ovsenek, Nick

doi:10.1002/bab.24

Cited by 5 publications

(6 citation statements)

References 42 publications

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“…3B ). In order to further characterize the biochemical properties of SpYY1 we bacterially expressed the protein and carried out purification via immobilized metal affinity chromatography followed by size exclusion chromatography using methods developed in our lab for the production of zinc finger proteins (previously described in 53 ). The resulting highly purified protein preparation (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In addition, the role of YY1 in embryonic development remains poorly understood. Given our previous extensive analysis of Xenopus YY1 40 , 42 – 44 , 53 , we sought to analyze the biochemical properties and subcellular localization of YY1 in the purple sea urchin Strongylocentrotus purpuratus , a major model organism in developmental biology. We have identified the YY1 homologue in S. purpuratus and named it SpYY1 .…”

Section: Discussionmentioning

confidence: 99%

“…Nuclei from oocytes were isolated according to the method of Thaler et al . 53 . Briefly, unfertilized eggs were collected as above by injection of potassium chloride solution and washed in ice-cold sea water.…”

Section: Methodsmentioning

confidence: 99%

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Conserved RNA binding activity of a Yin-Yang 1 homologue in the ova of the purple sea urchin Strongylocentrotus purpuratus

Belak

Ovsenek

Eskiw

2018

Sci Rep

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Yin-Yang 1 (YY1) is a highly conserved transcription factor possessing RNA-binding activity. A putative YY1 homologue was previously identified in the developmental model organism Strongylocentrotus purpuratus (the purple sea urchin) by genomic sequencing. We identified a high degree of sequence similarity with YY1 homologues of vertebrate origin which shared 100% protein sequence identity over the DNA- and RNA-binding zinc-finger region with high similarity in the N-terminal transcriptional activation domain. SpYY1 demonstrated identical DNA- and RNA-binding characteristics between Xenopus laevis and S. purpuratus indicating that it maintains similar functional and biochemical properties across widely divergent deuterostome species. SpYY1 binds to the consensus YY1 DNA element, and also to U-rich RNA sequences. Although we detected SpYY1 RNA-binding activity in ova lysates and observed cytoplasmic localization, SpYY1 was not associated with maternal mRNA in ova. SpYY1 expressed in Xenopus oocytes was excluded from the nucleus and associated with maternally expressed cytoplasmic mRNA molecules. These data demonstrate the existence of an YY1 homologue in S. purpuratus with similar structural and biochemical features to those of the well-studied vertebrate YY1; however, the data reveal major differences in the biological role of YY1 in the regulation of maternally expressed mRNA in the two species.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Conserved RNA binding activity of a Yin-Yang 1 homologue in the ova of the purple sea urchin Strongylocentrotus purpuratus

Belak

Ovsenek

Eskiw

2018

Sci Rep

Self Cite

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“…In spite of that the functional GO analysis shows that the cation binding proteins increased compared to the control. This group includes cadherin, phosphopantothenate-cysteine ligase as well as zinc finger protein, all of them purified by means of IMAC. Other metal ions such as cobalt, zinc, nickel, and aluminum can form complexes in the place of copper with some influence on protein interaction.…”

Section: Resultsmentioning

confidence: 99%

Widening and Diversifying the Proteome Capture by Combinatorial Peptide Ligand Libraries via Alcian Blue Dye Binding

et al. 2015

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Combinatorial peptide ligand libraries (CPLLs) tend to bind complex molecules such as dyes due to their aromatic, heterocyclic, hydrophobic, and ionic nature that may affect the protein capture specificity. In this experimental work Alcian Blue 8GX, a positively charged phthalocyanine dye well-known to bind to glycoproteins and to glucosaminoglycans, was adsorbed on a chemically modified CPLL solid phase, and the behavior of the resulting conjugate was then investigated. The control and dye-adsorbed beads were used to harvest the human urinary proteome at physiological pH, this resulting in a grand total of 1151 gene products identified after the capture. Although the Alcian Blue-modified CPLL incremented the total protein capture by 115 species, it particularly enriched some families among the harvested proteins, such as glycoproteins and nucleotide-binding proteins. This study teaches that it is possible, via the two combined harvest mechanisms, to drive the CPLL capture toward the enrichment of specific protein categories.

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“…Coomassie Blue R‐250 was used to detect the proteins after SDS–PAGE. Native‐PAGE (12% w/v acrylamide) was utilized to estimate the number of subunits in the protein . Egg white albumin (45 kDa) and bovine serum albumin (BSA; 67 kDa) were protein markers.…”

Section: Methodsmentioning

confidence: 99%

Improving the thermal stability of azoreductase from Halomonas elongata by introducing a disulfide bond via site‐directed mutagenesis

Nakhaee

Asad

Khajeh

et al. 2018

Biotech and App Biochem

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Azoreductases mainly reduce azo dyes, the largest class of colorants, to colorless aromatic amines. AzoH, a new azoreductase from the halophilic bacterium, Halomonas elongata, has been recently cloned and expressed in Escherichia coli. The aim of this study was to improve thermal stability of this enzyme by introducing new disulfide bonds. Since X-ray crystallography was not available, homology modeling and molecular dynamics was used to construct the enzyme three-dimensional structure. Potential disulfide bonds for increasing thermal stability were found using DIScover online software. Appropriate mutations (L49C/D108C) to form a disulfide bond were introduced by the Quik-Change method. Mutant protein expressed in E. coli showed increased thermal stability at 50 °C (increased half-life from 12.6 Min in AzoH to 26.66 Min in a mutated enzyme). The mutated enzyme could also tolerate 5% (w/v) NaCl and retained 30% of original activity after 24 H incubation, whereas the wild-type enzyme was completely inactivated. According to circular dichroism studies, the secondary structure was not altered by this mutation; however, a blue shift in intrinsic florescent graph revealed changes in the tertiary structure. This is the first study to improve thermal stability and salt tolerance of a halophilic azoreductase.

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Parameters for effective in vitro production of zinc finger nucleic acid‐binding proteins

Cited by 5 publications

References 42 publications

Conserved RNA binding activity of a Yin-Yang 1 homologue in the ova of the purple sea urchin Strongylocentrotus purpuratus

Conserved RNA binding activity of a Yin-Yang 1 homologue in the ova of the purple sea urchin Strongylocentrotus purpuratus

Widening and Diversifying the Proteome Capture by Combinatorial Peptide Ligand Libraries via Alcian Blue Dye Binding

Improving the thermal stability of azoreductase from Halomonas elongata by introducing a disulfide bond via site‐directed mutagenesis

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