Chemical synthesis and expression of a calmodulin gene designed for site-specific mutagenesis.
Chemical synthesis and expression of a calmodulin gene designed for site-specific mutagenesis.
复制标题
设计用于位点特异性诱变的钙调蛋白基因的化学合成和表达。
DOI：
10.1021/bi00340a020
复制
发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
Watterson,DM
中科院分区：
文献类型：
--
作者：
Roberts,DM;Crea,R;Malecha,M;Alvarado-Urbina,G;Chiarello,RH;Watterson,DM
材料和方法脱氧寡核苷酸。寡核苷酸的化学合成通过如Crea & Horn（1980）所述的磷酸三酯方法或通过如下所述的自动化亚磷酸三酯方法进行。寡核苷酸calmo-1-31（图1）是通过磷酸三酯法（Crea & Horn，1980）在纤维素载体上由二核苷酸试剂合成的。寡核苷酸calmo-32-61（图1）由二异丙基亚磷酰胺核苷酸（Matteucci &卡鲁瑟斯，1981）通过使用Alvarado-Urbina et al.（1981年）。在含有15 mL无水乙腈、0.6 mL无水2，6-二甲基吡啶和0.2 mL的反应混合物中，将5 H-（二甲氧基三苯甲基）-2H-脱氧核苷（1 mmol）转化为相应的二异丙基亚磷酰胺衍生物。缩写：pVUC-1（范德比尔特大学钙调蛋白1），由pKK 223 -3（PL Biochemicals; Amann et al.，1983）和合成的钙调蛋白基因; VU-1钙调蛋白，pVUC-1的表达产物; IPTG，异丙基/3-D-硫代半乳糖苷; Pth，苯基-硫代乙内酰脲; DTT，二硫苏糖醇; BSA，牛血清白蛋白; EGTA，乙二醇双（β-氨基乙基醚）-N，N，N-四乙酸;三甲基赖氨酸，N，N-二甲基赖氨酸; '.不N '-三甲基赖氨酸; calmo，钙调素;脱氧核糖核酸酶; EDTA，乙二胺四乙酸; NAD，烟酰胺腺嘌呤二核苷酸; PEG，聚（乙二醇）; Tris，三-（羟甲基）氨基甲烷; Tris-HCl，Tris盐酸盐。
Materials and MethodsDeoxyoligonucleotides. The chemical synthesis of oligo-nucleotides was performed by a phosphotriester method as described by Crea & Horn (1980) or by an automated phosphite triester method as described below. Oligonucleotides calmo-1-31 (Figure 1) were synthesized from dinucleotide reagents on a cellulose support by the phosphotriester method (Crea & Horn, 1980). Oligonucleotides calmo-32-61 (Figure1) were synthesized from diisopropylphosphoramidite nu-cleotides (Matteucci & Caruthers, 1981) by using the auto-mated stepwise addition protocol of Alvarado-Urbina et al.(1981). 5/-(Dimethoxytrityl)-2/-deoxynucleosides (1 mmol) were converted into the corresponding diisopropylphosphoramidite derivatives in reaction mixtures containing 15 mL of anhydrous acetonitrile, 0.6 mL of dry 2, 6-lutidine, and 0.2 mL1 Abbreviations: pVUC-1 (Vanderbilt University calmodulin 1), plasmid vector composed of pKK223-3 (PL Biochemicals; Amann et al., 1983) and the syntheticcalmodulin gene; VU-1 calmodulin, expressed product of pVUC-1; IPTG, isopropyl/3-D-thiogalactoside; Pth, phenyl-thiohydantoin; DTT, dithiothreitol; BSA, bovine serum albumin; EGTA, ethylene glycol bis (/3-aminoethyl ether)-iV, 7V, NyV-tetraacetic acid; tri-methyllysine, N.'. N'. N'-trimethyllysine; calmo, calmodulin; DNase, de-oxyribonuclease; EDTA, ethylenediaminetetraacetic acid; NAD, nico-tinamide adenine dinucleotide; PEG, poly (ethylene glycol); Tris, tris-(hydroxymethyl) aminomethane; Tris-HCl, Tris hydrochloride.