Soluble expression and characterization of a GFP-fused pea actin isoform (PEAc1)

• Published in: Cell research Vol. 14; no. 5; pp. 407 - 414
• Main Authors: Liu, Ai Xiao, Zhang, Shao Bin, Xu, Xiao Jing, Ren, Dong Tao, Liu, Guo Qin
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 01.10.2004; State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Science, China Agricultural University,Beijing 100094, China
• Subjects: 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; Nicotiana - cytology; Nicotiana - metabolism; Pisum sativum - chemistry; Prokaryotic Cells - metabolism; Protein Isoforms; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - metabolism; Recombinant Fusion Proteins - pharmacology; Time Factors; DNase I inhibition; polymerization; myosin Mg-ATPase activation; expression; actin isoform
• ISSN: 1001-0602; 1748-7838
• DOI: 10.1038/sj.cr.7290241

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.