Specific amino acid substitutions in bacterioopsin: Replacement of a restriction fragment in the structural gene by synthetic DNA fragments containing altered codons.
Specific amino acid substitutions in bacterioopsin: Replacement of a restriction fragment in the structural gene by synthetic DNA fragments containing altered codons.
复制标题
细菌视蛋白中的特定氨基酸取代：用含有改变密码子的合成 DNA 片段替换结构基因中的限制性片段。
DOI：
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发表时间：
1984
影响因子：
11.1
通讯作者：
Ala Tyr
中科院分区：
文献类型：
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作者：
Ser Glu;Ala Lys;Val Leu;Ala Ser;Thr Phe;Lys Val;Ala Glu;Ile Ala Gly;L. Phe;Ala Tyr
为了研究细菌视紫红质依赖光的质子转运机制，我们在基因中引入了单密码子改变，以便在蛋白质中产生以下特定的氨基酸替换：将酪氨酸 - 185替换为苯丙氨酸，脯氨酸 - 186替换为亮氨酸，色氨酸 - 189替换为苯丙氨酸，丝氨酸 - 193替换为丙氨酸，谷氨酸 - 194替换为谷氨酰胺。该策略涉及用含有修饰核苷酸序列的合成DNA双链体替换一个62碱基对的限制性片段。这需要在异亮氨酸 - 203处有一个独特的限制性位点（Xho I），它是通过寡核苷酸定向点突变产生的。与修饰的天然和突变限制性片段相对应的六个DNA双链体都是通过DNA连接酶催化连接化学合成的脱氧核糖寡核苷酸来制备的。使用合成DNA片段重建的细菌视蛋白表达质粒通过限制性分析和DNA序列测定进行了表征。开发了一种极其快速、高效且通用的用于纯化合成寡核苷酸和DNA片段的方法。
To study the mechanism of light-dependent proton translocation by bacteriorhodopsin, we have introduced single-codon changes in the gene so as to produce the following specific amino acid substitutions in the protein: Tyr-185 to Phe, Pro-186 to Leu, Trp-189 to Phe, Ser-193 to Ala, and Glu-194 to Gln. The strategy involved replacement of a 62-base-pair restriction fragment by synthetic DNA duplexes containing the modified nucleotide sequences. This required a unique restriction site (Xho I) at Ile-203 which was created by oligonucleotide-directed point mutagenesis. The six DNA duplexes corresponding to the modified native and mutant restriction fragments were all prepared by DNA ligase-catalyzed joining of chemically synthesized deoxyribooligonucleotides. The bacterioopsin expression plasmids reconstructed by using the synthetic DNA fragments were characterized by restriction analysis and DNA sequence determination. An extremely rapid, efficient, and general method for purification of the synthetic oligonucleotides and of DNA fragments was developed.