ARTICLES

Cell Research (2004); 14(5):407-414

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.

Received, Dec 8, 2003    Revised, May 28, 2004    Accepted, Jun 8, 2004

^*These authors contributed equally to this work.

Correspondence: Guo Qin Liu 86-10-62893438 (phone) 86-10-62891332 (fax) Liu@cau.edu.cn

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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^TM 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.

Keywords: actin isoform, polymerization, DNase I inhibition, myosin Mg-ATPase activation, expression.