Teparić Renata, Didak Blanka, Ščulac Elena, Mrša Vladimir
Laboratory of Biochemistry, Faculty of Food Technology and Biotechnology, University of Zagreb, 10000 Zagreb, Croatia.
J Gen Appl Microbiol. 2013;59(1):75-82. doi: 10.2323/jgam.59.75.
Genetic immobilization of the yeast RNase Rny1p was performed by creating a hybrid protein containing the signal sequence of the S. cerevisiae cell wall protein Ccw12p followed by the catalytic part of the Rny1p (amino acids 19 to 293) and additionally 73 amino acids of the Ccw12p including the GPI-anchoring signal. The construct was expressed in S. cerevisiae VMY5678 and the hybrid protein was secreted through the plasma membrane and incorporated into the cell wall through GPI-anchoring in the same way as the Ccw12p. Thus, it could be released from the wall by β-1,3-glucanase. It retained RNase activity with the optimal pH of about 9 and the optimal temperature at 60°C. It was significantly more stable than the wild type enzyme and retained activity at 50°C for at least 6 hours; at 60°C it maintained full activity for at least 4 h, and at 70°C it lost activity in about 2 h. No DNase activity of the Rny1/Ccw12p was detected. Yeast cells expressing the hybrid protein were successfully used instead of RNase A in a standard procedure for yeast chromosomal DNA preparation with the advantage of quick and easy quantitative removal of the RNase activity from the reaction mixture.
通过构建一种杂合蛋白来实现酵母核糖核酸酶Rny1p的基因固定化，该杂合蛋白包含酿酒酵母细胞壁蛋白Ccw12p的信号序列，随后是Rny1p的催化部分（第19至293个氨基酸），此外还包含Ccw12p的73个氨基酸，包括糖基磷脂酰肌醇（GPI）锚定信号。该构建体在酿酒酵母VMY5678中表达，杂合蛋白通过质膜分泌，并通过与Ccw12p相同的方式通过GPI锚定整合到细胞壁中。因此，它可以被β-1,3-葡聚糖酶从细胞壁上释放出来。它保留了核糖核酸酶活性，最佳pH约为9，最佳温度为60°C。它比野生型酶明显更稳定，在50°C下至少保留活性6小时；在60°C下它至少保持4小时的完全活性，在70°C下约2小时失去活性。未检测到Rny1/Ccw12p的脱氧核糖核酸酶活性。在酵母染色体DNA制备的标准程序中，成功地使用表达杂合蛋白的酵母细胞代替核糖核酸酶A，其优点是能快速、轻松地从反应混合物中定量去除核糖核酸酶活性。
