PMID:15538972
| Citation |
Liu, AX, Zhang, SB, Xu, XJ, Ren, DT and Liu, GQ (2004) Soluble expression and characterization of a GFP-fused pea actin isoform (PEAc1). Cell Res. 14:407-14 |
|---|---|
| Abstract |
A pea actin isoform PEAc1 with green fluorescent protein (GFP) fusion to its C-terminus and His-tag to its N-terminus, was expressed in prokaryotic cells in soluble form, and highly purified with Ni-Chelating Sepharose Fast Flow column. The purified fusion protein (PEAc1-GFP) efficiently inhibited DNase I activities before polymerization, and activated the myosin Mg-ATPase activities after polymerization. The PEAc1-GFP also polymerized into green fluorescent filamentous structures with a critical concentration of 0.75 uM. These filamentous structures were labeled by TRITC-phalloidin, a specific agent for staining actin microfilaments, and identified as having 9 nm diameters by negative staining. These results indicated that PEAc1 preserved the essential characteristics of actin even with His-tag and GFP fusion, suggesting a promising potential to use GFP fusion protein in obtaining soluble plant actin isoform to analyze its physical and biochemical properties in vitro. The PEAc1-GFP was also expressed in tobacco BY2 cells, which offers a new pathway for further studying its distribution and function in vivo. |
| Links |
PubMed Online version:10.1038/sj.cr.7290241 |
| Keywords |
Actins/biosynthesis; Actins/metabolism; Actins/pharmacology; Animals; Ca(2+) Mg(2+)-ATPase/drug effects; Ca(2+) Mg(2+)-ATPase/metabolism; Deoxyribonuclease I/antagonists & inhibitors; Deoxyribonuclease I/metabolism; Green Fluorescent Proteins/biosynthesis; Green Fluorescent Proteins/metabolism; Green Fluorescent Proteins/pharmacology; Myosins/metabolism; Peas/chemistry; Prokaryotic Cells/metabolism; Protein Isoforms; Recombinant Fusion Proteins/biosynthesis; Recombinant Fusion Proteins/metabolism; Recombinant Fusion Proteins/pharmacology; Time Factors; Tobacco/cytology; Tobacco/metabolism |
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