Cloning, soluble expression, rapid purification and characterization of human Cofilin1

Lu, Jie; Xiang, Yangfei; Zhang, Jiaxuan; Ju, Huai-Qiang; Chen, Zhenping; Wang, Qiaoli; Chen, Wei; Peng, Xinlei; Han, Bo; Wang, Yifei

doi:10.1016/j.pep.2012.01.002

Cited by 6 publications

(3 citation statements)

References 31 publications

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“…Protein was expressed and purified as described previously. Briefly, TaADF7 in pET‐41a (+) was expressed in E. coli strain BL21, and after induction with isopropyl‐β‐ d ‐thiogalactoside, protein was extracted and purified as described previously (Moseley et al ., ; Lu et al ., ). Protein was pooled and dialyzed into KMEI buffer (Mason et al ., ).…”

Section: Methodsmentioning

confidence: 97%

TaADF7, an actin‐depolymerizing factor, contributes to wheat resistance against Puccinia striiformis f. sp. tritici

et al. 2014

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SUMMARYThe actin cytoskeleton is involved in plant defense responses; however, the role of the actin-depolymerizing factor (ADF) family, which regulates actin cytoskeletal dynamics, in plant disease resistance, is largely unknown. Here, we characterized a wheat (Triticum aestivum) ADF gene, TaADF7, with three copies located on chromosomes 1A, 1B, and 1D, respectively. All three copies encoded the same protein, although there were variations in 19 nucleotide positions in the open reading frame. Transcriptional expression of the three TaADF7 copies were all sharply elevated in response to avirulent Puccinia striiformis f. sp. tritici (Pst) infection, with similar expression patterns. TaADF7 regulated the actin cytoskeletal dynamics by targeting the actin cytoskeleton to execute actin binding/severing activities. When the TaADF7 copies were all silenced by virus-induced gene silencing, the growth of Pst hypha increased and sporadic urediniospores were observed, as compared with control plants, upon inoculation with avirulent Pst. In addition, the accumulation of reactive oxygen species (ROS) and the hypersensitive response (HR) were greatly weakened, whereas cytochalasin B partially rescued the HR in TaADF7 knock-down plants. Together, these findings suggest that TaADF7 is likely to contribute to wheat resistance against Pst infection by modulating the actin cytoskeletal dynamics to influence ROS accumulation and the HR.

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Section: Methodsmentioning

confidence: 97%

TaADF7, an actin‐depolymerizing factor, contributes to wheat resistance against Puccinia striiformis f. sp. tritici

et al. 2014

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“…Cells were grown in Luria-Bertani (LB) media and puried by nickel-affinity chromatography as described. [35][36][37][38] Briey, bacterial cells were grown in LB media containing 50 mg mL −1 kanamycin at 37 °C with shaking to an OD 600 of 0.6-1.0, expression was induced with 1 mM IPTG and the culture was incubated while shaking for 5 h. Cells were then harvested by centrifugation at 4000 rpm for 20 min and resuspended in buffer A (20 mM Tris-HCl (pH 8.0) and 250 mM NaCl) with 10 mM imidazole, 0.1% v/v Triton X-100 and 1 tablet of EDTA-free protease inhibitor (Roche). The cell pellets from 4 L of culture were lysed by sonication on ice and centrifuged at 12 500 rpm for 40 min at 4 °C.…”

Section: Expression and Purication Of Selected Protein Partnersmentioning

confidence: 99%

Drug repurposing screens identify compounds that inhibit α-synuclein oligomers' membrane disruption and block antibody interactions

et al. 2023

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“…In each case, plasmids were transformed into the E. coli strain BL21 (DE3) cells separately for overexpression. Cells were grown in Luria-Bertani (LB) media and purified by nickel-affinity chromatography as described [28][29][30][31] . Briefly, bacterial cells were grown in LB media containing 50 µg/ml kanamycin at 37°C with shaking to an OD600 of 0.6-1.0, expression was induced with 1 mM IPTG and the culture was incubated while shaking for 5 h. Cells were then harvested by centrifugation at 4000 rpm for 20 min and re-suspended in buffer A (20 mM Tris-HCl (pH 8.0) and 250 mM NaCl) with 10 mM imidazole, 0.1% v/v Triton X-100 and 1 tablet of EDTA-free protease inhibitor (Roche).…”

Section: Expression and Purification Of Selected Protein Partnersmentioning

confidence: 99%

Drug repurposing screens identifies compounds that inhibit α-synuclein oligomers’ membrane disruption and block antibody interactions

Somavarapu

Kleijwegt

Nagaraj

et al. 2022

Preprint

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Small soluble oligomers of the protein α-synuclein (αSO) have been linked to disruptions in neuronal homeostasis, contributing to the development of Parkinson's Disease (PD). While this makes αSO an obvious drug target, the development of effective therapeutics against αSO are challenged by its low abundance and structural and morphological complexity. Here we employ two different approaches to neutralize toxic interactions made by αSOs with different cellular components. Firstly, we use available data to identify four neuronal proteins as likely candidates for αSO interactions, namely Cfl1, Uchl1, Sirt2 and SerRS. However, despite promising results when immobilized, all 4 proteins only bind weakly to αSO in solution in microfluidic assays, making them inappropriate for screening. In contrast, the formation of stable contacts formed between αSO and vesicles consisting of anionic lipids not only mimics a likely biological role of αSO but also provided a platform to screen two small molecule libraries for disruptors of these contacts. Of the 11 leads obtained in this way, 2 significantly impaired αSO contacts with other proteins in a sandwich ELISA assay using αSO-binding monoclonal antibodies and nanobodies. In addition, 5 of these leads suppressed α-synuclein amyloid formation. Thus a repurposing screening that directly targets a key culprit in PD pathogenesis shows therapeutic potential.

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Cloning, soluble expression, rapid purification and characterization of human Cofilin1

Cited by 6 publications

References 31 publications

TaADF7, an actin‐depolymerizing factor, contributes to wheat resistance against Puccinia striiformis f. sp. tritici

TaADF7, an actin‐depolymerizing factor, contributes to wheat resistance against Puccinia striiformis f. sp. tritici

Drug repurposing screens identify compounds that inhibit α-synuclein oligomers' membrane disruption and block antibody interactions

Drug repurposing screens identifies compounds that inhibit α-synuclein oligomers’ membrane disruption and block antibody interactions

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