CAJ | 학술논문

本研究将AF1514蛋白的重组基因转化到大肠杆菌(E.coli BL21)并通过在12℃条件下的诱导表达产生了大量的目的蛋白.用镍柱亲和层析和分子筛Superdex-75凝胶过滤层析的两步蛋白纯化方法进一步纯化蛋白AF1514后,获得纯度较高的蛋白(纯度＞95%).纯化的母体蛋白和甲基化处理后的蛋白衍生物分别用悬滴汽相扩散法在16℃结晶.初步筛选所用的400多种结晶条件中,适合晶体生长的条件只有3种,其中最佳晶体生长条件溶液的成分包含0.1mol/L醋酸钠,pH 5.0,0.1mol/L氯化钠和8%～14%(W/V)2-甲基2,4-戊二醇.蛋白晶体属于四方晶系,初步X-射线衍射分辨率为2.09A°.
본연구장AF1514단백적중조기인전화도대장간균(E.coli BL21)병통과재12℃조건하적유도표체산생료대량적목적단백.용얼주친화층석화분자사Superdex-75응효과려층석적량보단백순화방법진일보순화단백AF1514후,획득순도교고적단백(순도＞95%).순화적모체단백화갑기화처리후적단백연생물분별용현적기상확산법재16℃결정.초보사선소용적400다충결정조건중,괄합정체생장적조건지유3충,기중최가정체생장조건용액적성분포함0.1mol/L작산납,pH 5.0,0.1mol/L록화납화8%～14%(W/V)2-갑기2,4-무이순.단백정체속우사방정계,초보X-사선연사분변솔위2.09A°.
Archeoglobus fulgidus DSM 4304 genome was transformed and overproduced in Escherichia coli(E.coli BL21)at 12℃and the protein was purified to 95%purity by Ni2+-affinity colunm and Superdex-75 gel filtration.Crystals ative and methylated proteins were obtained by the hanging-drop vapour-diffusion method at 16℃under three conditions among the wide range of screened conditions(＞400),but principally from the solution containing 0.1mol/L sodium acetate pH 5.0,0.1mol/L sodium chloride,and 8%～14%(W/V)2-methyl-2,4-dipentaneol (MPD).The crystal is tetragonal in shape and diffraction data to 2.09A° are detected by X-ray detector.