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 purifyeach actin isoform in sufficient quantities...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 purifyeach actin isoform in sufficient quantities for analysing itsphysicochemical properties. In the present study, apea(pisum Sativum L.)actin isoform (PEAc1)fused to His-tag at its amino terminus and GFP(green fluorescent protein)atits Carboxyl terminus were expressed in E. coli in inclusionbodies. The fusion protein (PEAc1-GFP)was highly purifiedwith the yield of above 2 mg/L culture by dissolving inclu-sions 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 andinhibit the latter抯 enzyme activity. PEAc1-GFP could po-lymerise into green fluorescent filamentous structures(F-PEAc1-GFP),which could be labelled byTRITC-phalloidin,a specific agent for observing microfila-ments. The PEAc1-GFP polymerlzation curve was identicalwith that of chicken skeletal muscle actin. The critical con-centration for PEAc1-Gfp to polymerise into filaments is 0.24 μmol/L.The F-PEAc1-GFP could stimulate myosinMg-ATPase activity in a protein concentration dependantmanner (about 4 folds at 1 mg/mL F-PEAc1-GFP). The re-sults above show that the PEAc1 fused to GFP retained theassembly characteristic of actin, indicating that gene fusion,prokaryotic expression, denaturation and renaturation,andaffinity chromatography is a useful strategy for obtainingplant actin isoform proteins in a large amount.展开更多
文摘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 purifyeach actin isoform in sufficient quantities for analysing itsphysicochemical properties. In the present study, apea(pisum Sativum L.)actin isoform (PEAc1)fused to His-tag at its amino terminus and GFP(green fluorescent protein)atits Carboxyl terminus were expressed in E. coli in inclusionbodies. The fusion protein (PEAc1-GFP)was highly purifiedwith the yield of above 2 mg/L culture by dissolving inclu-sions 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 andinhibit the latter抯 enzyme activity. PEAc1-GFP could po-lymerise into green fluorescent filamentous structures(F-PEAc1-GFP),which could be labelled byTRITC-phalloidin,a specific agent for observing microfila-ments. The PEAc1-GFP polymerlzation curve was identicalwith that of chicken skeletal muscle actin. The critical con-centration for PEAc1-Gfp to polymerise into filaments is 0.24 μmol/L.The F-PEAc1-GFP could stimulate myosinMg-ATPase activity in a protein concentration dependantmanner (about 4 folds at 1 mg/mL F-PEAc1-GFP). The re-sults above show that the PEAc1 fused to GFP retained theassembly characteristic of actin, indicating that gene fusion,prokaryotic expression, denaturation and renaturation,andaffinity chromatography is a useful strategy for obtainingplant actin isoform proteins in a large amount.
