Cloning, soluble expression, rapid purification and characterization of human Cofilin1
Biomedicine Research and Development Center, Guangdong Provincial Key Laboratory of Bioengineering Medicine, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou 510632, China. Protein Expression and Purification
(Impact Factor: 1.7).
03/2012; 82(1):186-91. DOI: 10.1016/j.pep.2012.01.002
Cofilin1 is an actin-binding protein that plays a critical role in the regulation of actin cytoskeleton and consequently affects various physiological processes. In this study, the human Cofilin1 cDNA was cloned into the expression vector pET-28a(+) with a 6 × His tag and expressed as soluble protein in Escherichia coli BL21(DE3). Approximately 78 mg of Cofilin1, which showed high activity as determined by native PAGE, could be purified from each liter of LB medium by His-tag affinity chromatography and gel filtration. Further, high-titer IgG against Cofilin1 was positively detected after immunization in rabbits and the polyclonal antibodies were purified and identified. Together, this report provides the first protocol to efficiently obtain human Cofilin1 with high biological activity and immunogenicity using E. coli BL21 (DE3) expression system.
Available from: Ralf T. Voegele
- "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-b-D-thiogalactoside, protein was extracted and purified as described previously (Moseley et al., 2006; Lu et al., 2012). Protein was pooled and dialyzed into KMEI buffer (Mason et al., 2011). "
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ABSTRACT: The 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 region. 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, when inoculated with avirulent Pst. In addition, reactive oxygen species (ROS) accumulation and hypersensitive response (HR) were greatly weakened, whereas cytochalasin B partially rescued the HR in TaADF7-knockdown plants. Together, these findings suggest that TaADF7 likely contributes to wheat resistance against Pst infection by modulating the actin cytoskeletal dynamics to influence ROS accumulation and the HR. This article is protected by copyright. All rights reserved.
The Plant Journal 02/2014; 78(1). DOI:10.1111/tpj.12457 · 5.97 Impact Factor
Available from: Kaio Kitazato
- "Cells were lysed in RIPA buffer (Beyotime, P0013B) containing 1% PMSF (Beyotime, ST506) and clarified by centrifugation at 15,000Âg for 15 min at 4 °C. The protein concentration in the supernatant was measured using the BCA protein assay kit (Beyotime , P0010) . The cell lysates were mixed with 5Â SDS–PAGE buffer (Beyotime, Jiangsu, China) and boiled for 5 min. "
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ABSTRACT: Autophagy plays a crucial role in a wide array of physiological processes. To uncover the complex regulatory networks and mechanisms underlying basal autophagy, we performed a quantitative proteomics analysis of autophagy-deficient mouse embryonic fibroblast cells (MEFs) using iTRAQ labeling coupled with on-line 2D LC/MS/MS. We quantified a total of 1,234 proteins and identified 114 proteins that were significantly altered (90% confidence interval), including 48 up-regulated proteins and 66 down-regulated proteins. We determined that F-actin was disassembled in autophagy-deficient Atg7(-/-) MEFs. Treatment of the WT MEFs with cytochalasin D (CD), which induces F-actin depolymerization, significantly induced autophagosome formation. However, treatment with cytochalasin D also increased the protein level of p62 under starvation conditions, suggesting that depolymerization of F-actin impaired autophagosome maturation and that the intact F-actin network is required for basal and starvation-induced autophagy. Our results demonstrate a close relationship between F-actin and autophagy and provide the basis for further investigation of their interactions.
Biochemical and Biophysical Research Communications 07/2013; 437(3). DOI:10.1016/j.bbrc.2013.06.111 · 2.30 Impact Factor
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ABSTRACT: X-ray crystallography for the determination of three-dimensional structures of protein macromolecules represents an important tool in function assignment of uncharacterized proteins. However, crystallisation is often difficult to achieve. A protein sample fully characterized in terms of dispersity may increase the likelihood of successful crystallisation by improving the predictability of the crystallisation process. To maximize the probability of crystallisation of a novel mouse macrophage protein (rMMP), target molecule was characterized and refined to improve monodispersity. Addition of MgCl2 at low concentrations resolves the rMMP into a monodisperse solution, and finally successful crystallization of rMMP was achieved. The effect of MgCl2 was studied using gel filtration chromatography and dynamic light scattering.
Molecular and Cellular Biochemistry 01/2014; 389(1). DOI:10.1007/s11010-013-1934-x · 2.39 Impact Factor
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