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Please use this identifier to cite or link to this item: http://ir.ncue.edu.tw/ir/handle/987654321/16766

Title: 嗜熱性藍綠茵Synechococcus lividus之蛋白質去磷酸酪:選殖、表現、純他及活性分析
Cloning, Expression, Purification and Activity Assay of the Protein Phosphatase from a Thermophilic Cyanobacterium Synechococcus Lividus
Authors: 蕭蓉禎;王瑋龍;劉婉琳;蔡顯麞;林宗岐;溫育德
Contributors: 生物學系
Keywords: 嗜熱性藍綠菌;蛋白質去磷酸醋;表現;活性分析
Thermophilic cyanobacterium;Protein phosphatase;Expression;Activity assay
Date: 2011-12
Issue Date: 2013-06-05T08:46:19Z
Publisher: 中國生物學會
Abstract: 利用聚合酶連鎖反應技術,選殖嗜熱性藍綠菌Synechococcus lividus之蛋白質去磷酸醋,此基因全長為240個胺基酸,與protein phosphatase 2C(PP2C)非常相似。因而將此基因命名syPP2C。胺基酸序列比對的結果顯示syPP2C與Thermosynechococcus elongatus BP-1的PP2C較為相似(95%相同度),而與其它藻類的PP2C較為不同(<60%相同度)。將syPP2C選殖至表現載體pET21a,並送入大腸桿菌E. coli BL21(DE3)進行蛋白質表現,結果顯示重組6*His_syPP2C蛋白可以在菌體內,以可溶狀態(soluble form)大量表現,進一步以Ni(上標 +)管柱純化後,進行重組6*His_syPP2C蛋白的酵素特性分析,結果如下:(一)重組6*His_syPP2C蛋白的最適反應溫度為55°C;(二)Mn(上標 2+)或Mg(上標 2+)離子皆可增強重組伊6*His_syPP2C蛋白的酵素活性;(三)重組6*His_syPP2C蛋白的酵素活性並不會受到去磷酸酪抑制劑okadaic acid的影響,但會被EDTA所抑制。
A gene similar to protein phosphatase 2C- type phosphatase (PP2C) was cloned from a thermophilic cyanobacterium Synechococcus lividus using the polymerase chain reaction technique. This gene named syPP2C, encodes 240 amino acids. At amino acid level, syPP2C is the most similar to Thermosynechococcus elongatus BP-1 PP2C (95%), but less similar (< 60%) to other PP2C of different algae. The recombinant 6*His_syPP2C was successfully overexpressed in E. coli as a soluble form, and purified by Ni(superscript +) affinity chromatography. Phosphatase activity assay indicated that (1) the optimal temperature of recombinant 6*His_syPP2C was 55℃; (2) the presence of Mn(superscript 2+) or Mg(superscript 2+) could enhance the enzymatic activity of 6*His_syPP2C; (3) the enzymatic activity of 6*His_syPP2C could be inhibited by EDTA, but not by okadaic acid.
Relation: 生物科學, 53(2): 51-64
Appears in Collections:[生物學系] 期刊論文

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