Prokaryotic expression and characterization of a pea actin isoform (PEAcl) fused to GFP

Actins widely exist in eukaryotic cells and play important roles in many living activities. As there are many kinds of actin isoforms in plant cells, it is difficult to purify each actin isoform in sufficient quantities for analyzing its physicochemical properties. In the present study, a pea(Pisum Sativum L.) actin isoform (PEAc1) fused to His-tag at its amino terminus and GFP (green fluorescent protein) at its Carboxyl terminus were expressed in E. Coli in inclusion bodies. The fusion protein (PEAc1-GFP) was highly purified with the yield of above 2 mg/L culture by dissolving inclusions in 8 mol/L urea, renaturing by dialysis in a gradient of urea, and affinity binding to Ni-resin. The purified mono meric PEAc1-GFP could efficiently bind on Dnase Ⅰ and inhibit the latter's enzyme activity. PEAc1-GFP could polymerize into green fluorescent filamentous structures (F-PEAc1-GFP), which could be labeled by TRITC-phalloidin, a specific agent for observing microfilaments. The PEAc1-GFP polymerization curve was identical with that of chicken skeletal muscle actin. The critical concentration for PEAc1-GFP to polymerize into filaments is 0.24 μmol/L. The F-PEAc1-GFP could stimulate myosin Mg-ATPase activity in a protein concentration dependant manner (about 4 folds at1 mg/mL F-PEAc1-GFP). The results above show that the PEAcl fused to GFP retained the assembly characteristic of actin, indicating that gene fusion, prokaryotic expression,denaturation and renaturation, and affinity chromatography is a useful strategy for obtaining plant actin isoform proteins in a large amount.

作 者： Zhang Shaobin REN Dongtao XU Xiaojing LIU Guoqin   作者單位： State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Science, China Agricultural University, Beijing 100094,China  刊 名： 科學(xué)通報(bào)(英文版)  SCI 英文刊名： CHINESE SCIENCE BULLETIN  年，卷(期)： 2004 49(9)  分類號(hào)： Q94  關(guān)鍵詞： actin   isoform   prokaryotic expression   purification   physicochemical properties  

【Prokaryotic expression and character】相關(guān)文章：

Character Analysis of Scarlett and Melanie04-26

Investigation of the Aerodynamic Character in Turbine Cascades with Aft-loaded Profile04-26

Induction of defence gene expression by oligogalacturonic acid requires increases in both cytosolic calcium and hydrogen04-26

Induction of defence gene expression by oligogalacturonic acid requires increases in both cytosolic calcium and hydrogen04-26

Down-Regulated Expression of RACK1 Gene by RNA Interference Enhances Drought Tolerance in Rice04-26

Detection of CD2 expression in chicken hematogenic embryo yolk sac lymphoid cells prior to thymus genesis04-26