Penna Thereza Christina Vessoni, Ishii Marina, Junior Adalberto Pessoa, Cholewa Olivia
Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Science, University of São Paulo, Rua Antonio de Macedo Soares, 452, 04607-000, São Paulo/SP, Brazil.
Appl Biochem Biotechnol. 2004 Spring;113-116:469-83. doi: 10.1385/abab:114:1-3:469.
The thermal stability of the recombinant green fluorescent protein (GFPuv) expressed by Escherichia coli cells and isolated by three-phase partitioning extraction with hydrophobic interaction chromatography was studied. The GFPuv (3.5-9.0 microg of GFPuv/mL) was exposed to various pH conditions (4.91-9.03) and temperatures (75-95 degrees C) in the 10 mM buffers: acetate (pH 5.0-7.0), phosphate (pH 5.5-8.0), and Tris-HCl (pH 7.0-9.0). The extent of protein denaturation (loss of fluorescence intensity) was expressed in decimal reduction time (D-value), the time exposure required to reduce 90% of the initial fluorescence intensity of GFPuv. For pH 7.0 to 8.0, the thermostability of GFPuv was slightly greater in phosphate buffer than in Tris-HCl. At 85 degrees C, the D-values (pH 7.1-7.5) ranged from 7.24 (Tris-HCl) to 13.88 min (phosphate). The stability of GFPuv in Tris-HCl (pH >8.0) was constant at 90 and 95 degrees C, and the D-values were 7.93 (pH 8.38-8.92) and 6.0 min (pH 8.05-8.97), respectively. The thermostability of GFPuv provides the basis for its potential utility as a fluorescent biologic indicator to assay the efficacy of moist-heat treatments at temperatures lower than 100 degrees C.
研究了大肠杆菌细胞表达并通过疏水相互作用色谱三相分配萃取分离的重组绿色荧光蛋白（GFPuv）的热稳定性。将GFPuv（3.5 - 9.0微克GFPuv/毫升）置于10 mM缓冲液（醋酸盐，pH 5.0 - 7.0；磷酸盐，pH 5.5 - 8.0；Tris - HCl，pH 7.0 - 9.0）中，暴露于各种pH条件（4.91 - 9.03）和温度（75 - 95℃）下。蛋白质变性程度（荧光强度损失）用十进制减少时间（D值）表示，即降低GFPuv初始荧光强度90%所需的暴露时间。在pH 7.0至8.0时，GFPuv在磷酸盐缓冲液中的热稳定性略高于Tris - HCl缓冲液。在85℃时，D值（pH 7.1 - 7.5）范围为7.24分钟（Tris - HCl）至13.88分钟（磷酸盐）。在90℃和95℃时，GFPuv在Tris - HCl（pH >8.0）中的稳定性恒定，D值分别为7.93分钟（pH 8.38 - 8.92）和6.0分钟（pH 8.05 - 8.97）。GFPuv的热稳定性为其作为荧光生物指示剂在低于100℃的湿热处理效果检测中的潜在应用提供了基础。
