Lin S F, Lin S W, Hsu T Y, Liu M Y, Chen J Y, Yang C S
Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei, Republic of China.
Virology. 1994 Feb 15;199(1):223-7. doi: 10.1006/viro.1994.1115.
A cDNA coding for the Epstein-Barr virus (EBV) alkaline deoxyribonuclease (DNase) was expressed in Escherichia coli using the T7 phage system designed to allow expression of potentially lethal proteins. Induction of protein synthesis from the gene yielded a peptide with a molecular weight of approximately 52 kDa, consistent with the predicted open reading frame of EBV BGLF5. A high level of nuclease activity was detected in crude cell extracts, and the activity was neutralized by sera from nasopharyngeal carcinoma patients with high titers of anti-DNase antibodies. A series of deletion clones truncated at the amino or carboxyl terminus was constructed and expressed to define the regions responsible for the nuclease activity of EBV DNase. All the mutated molecules lost their activities even though their expression levels were comparable to that of the full-length DNase. To determine the exact role of the amino terminus of EBV DNase, mutants with small deletions were expressed. While three mutants with deletions of amino acid residues 11-30, 16-28, or 23-28 failed to show any detectable nuclease activities, one mutant in which the first 8 amino acids were replaced by the first 12 amino acid residues of the T7 major capsid protein contributed by the vector was enzymatically active. To further define the importance of the amino-terminal region, full-length DNase with point mutations was generated among residues 23-29 by site-directed mutagenesis and expressed in the same system. Assays of the DNase activity of these mutants revealed that the mutation of residue 29 was fully active, and mutations in 24-27 retained 50% activity. Nevertheless, the mutation at residue 23 resulted in a complete loss of activity and the mutation at residue 28 resulted in loss of 70% activity. These results suggest the biological importance of the amino terminus of the EBV DNase, especially residues 23-28.
利用旨在允许表达潜在致死性蛋白质的T7噬菌体系统，在大肠杆菌中表达了编码爱泼斯坦 - 巴尔病毒（EBV）碱性脱氧核糖核酸酶（DNase）的cDNA。从该基因诱导蛋白质合成产生了一种分子量约为52 kDa的肽，这与EBV BGLF5预测的开放阅读框一致。在粗细胞提取物中检测到高水平的核酸酶活性，并且来自具有高滴度抗DNase抗体的鼻咽癌患者的血清可中和该活性。构建并表达了一系列在氨基或羧基末端截短的缺失克隆，以确定负责EBV DNase核酸酶活性的区域。所有突变分子均失去活性，尽管它们的表达水平与全长DNase相当。为了确定EBV DNase氨基末端的确切作用，表达了具有小缺失的突变体。虽然三个缺失氨基酸残基11 - 30、16 - 28或23 - 28的突变体未显示任何可检测到的核酸酶活性，但一个其中前8个氨基酸被载体贡献的T7主要衣壳蛋白的前12个氨基酸残基取代的突变体具有酶活性。为了进一步确定氨基末端区域的重要性，通过定点诱变在残基23 - 29之间产生了具有点突变的全长DNase，并在同一系统中表达。对这些突变体的DNase活性测定表明，残基29的突变体完全有活性，24 - 27位的突变保留了50%的活性。然而，残基23处的突变导致活性完全丧失，残基28处的突变导致70%的活性丧失。这些结果表明EBV DNase氨基末端的生物学重要性，尤其是残基23 - 28。
