Soluble expression and characterization of a GFP-fused pea actin isoform (PEAc1).
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.
Liu
Ai Xiao
AX
State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Science, China Agricultural University, Beijing 100094, China. Liu@cau.edu.cn
Zhang
Shao Bin
SB
Xu
Xiao Jing
XJ
Ren
Dong Tao
DT
Liu
Guo Qin
GQ
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
D000199:Actins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D003850:Deoxyribonuclease I; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins