CAJ | 학술논문

丙氨酸脱氢酶(alanine dehydrogenase,Ald)是一种NAD+依赖性的氨基酸脱氢酶,能可逆地催化丙氨酸氧化脱氨生成丙酮酸和氨.实验以假坚强芽孢杆菌的丙氨酸脱氢酶为研究对象,将目的基因克隆到 pET-22,b(+)原核表达载体上,并在大肠杆菌中完成蛋白的高效表达.通过镍离子亲和层析、离子交换层析和凝胶过滤层析等纯化方法,得到了高纯度的目的蛋白.利用气相扩散法对目的蛋白进行结晶,最终得到分辨率为0.31,nm的蛋白晶体.
병안산탈경매(alanine dehydrogenase,Ald)시일충NAD+의뢰성적안기산탈경매,능가역지최화병안산양화탈안생성병동산화안.실험이가견강아포간균적병안산탈경매위연구대상,장목적기인극륭도 pET-22,b(+)원핵표체재체상,병재대장간균중완성단백적고효표체.통과얼리자친화층석、리자교환층석화응효과려층석등순화방법,득도료고순도적목적단백.이용기상확산법대목적단백진행결정,최종득도분변솔위0.31,nm적단백정체.
Alanine dehydrogenase(Ald)is a NAD+-dependent amino acid dehydrogenase. It catalyzes the reversible oxida-tive deamination of L-alanine to pyruvate and ammonia. An Ald gene from Bacillus pseudofirmus was cloned into the pro-karyotic expression vector pET-22,b(+)and the Ald protein was overexpressed inEscherichia coli. The Ald protein was purified by Ni2+-chelating affinity chromatography,anion-exchange chromatography and gel filtration chromatography. Crystals were grown with the vapour-diffusion method and diffracted to 0.31,nm resolution.