Volume 14, No 5, Oct 2004

ISSN: 1001-0602 
EISSN: 1748-7838 2018 
impact factor 17.848* 
(Clarivate Analytics, 2019)

Volume 14 Issue 5, October 2004: 407-414

ORIGINAL ARTICLES

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

Ai Xiao LIU*, Shao Bin ZHANG*, Xiao Jing XU, Dong Tao REN, Guo Qin LIU**

State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Science, China Agricultural University, Beijing 100094, China. Correspondence: Guo Qin Liu(Liu@cau.edu.cn)

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 SepharoseTM 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 µM. 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 obtainning 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.

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