title: Soluble expression and characterization of a GFP-fused pea actin isoform (PEAc1).
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.
authors.affiliation: State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Science, China Agricultural University, Beijing 100094, China. Liu@cau.edu.cn
authors.forename: Ai Xiao
authors.initials: AX
authors.lastname: Liu
authors.forename: Shao Bin
authors.initials: SB
authors.lastname: Zhang
authors.forename: Xiao Jing
authors.initials: XJ
authors.lastname: Xu
authors.forename: Dong Tao
authors.initials: DT
authors.lastname: Ren
authors.forename: Guo Qin
authors.initials: GQ
authors.lastname: Liu
mesh_terms: D000199:Actins / Q000096:biosynthesis* / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000187:drug effects / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000037:antagonists & inhibitors / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000378:metabolism* / Q000494:pharmacology; D009218:Myosins / Q000378:metabolism; D018532:Pisum sativum / Q000737:chemistry*; D011387:Prokaryotic Cells / Q000378:metabolism; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism / Q000494:pharmacology; D013997:Time Factors; D014026:Nicotiana / Q000166:cytology / Q000378:metabolism
chemical_list: D000199:Actins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D003850:Deoxyribonuclease I; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins