獲得蛋白質的晶體結構的第一個瓶頸,就是製備大量純化的蛋白質(>10mg),其濃度通常在10mg/ml以上,並以此為基礎進行結晶條件的篩選。運用重組基因的技術,將特定基因以選殖(clone)的方式嵌入表現載體(expression vector)內,此一載體通常具有易於調控的特性。之後再將帶有特定基因的載體送入可快速生長的菌體中,如大腸杆菌(Escherichia coli),在zai菌jun體ti快kuai速su生sheng長chang的de同tong時shi,也ye大da量liang生sheng產chan表biao現xian載zai體ti上shang的de基ji因yin所suo解jie譯yi出chu之zhi蛋dan白bai質zhi。一yi般ban而er言yan純chun度du越yue高gao的de蛋dan白bai質zhi比bi較jiao有you機ji會hui形xing成cheng晶jing體ti,因yin此ci純chun化hua蛋dan白bai質zhi的de步bu驟zhou就jiu成cheng為wei一yi個ge重zhong要yao的de決jue定ding因yin素su。
zaiqudegaochundudedanbaizhirongyehou,jiexialaijiushijingtidepeiyang。danbaizhijingtiyuqitahuahewujingtidexingchengleisi,shizaibaoherongyezhongmanmanchanshengde,meiyizhongdanbaizhiyangjingdetiaojianjieyousuochayi,yingxiangjingtixingchengdebianlianghenduo,baohanhuaxueshangdebianliang,rusuanjiandu、沉澱劑種類、離子濃度、蛋白質濃度等;物理上的變數,如溶液達成過飽和狀態的速率、溫度等;及生化上的變數,如蛋白質所需的金屬離子或抑製劑、蛋白質的聚合狀態、dengdiandiandeng,jieshiyangjingshideceshitiaojian。jiezhimuqianweizhi,bingwuyitaolilunkeyiyucejiejingdetiaojian,suoyibixubuduanceshigezhongyangjingrongyedezuhehou,caikenengdedaoyikewanmeidedanyijingti。
蛋白質晶體的培養,通常是利用氣相擴散法(Vapor Diffusion Method)的原理來達成;也就是將含有高濃度的蛋白質(10~50mg/ml)溶rong液ye加jia入ru適shi當dang的de溶rong劑ji,慢man慢man降jiang低di蛋dan白bai質zhi的de溶rong解jie度du,使shi其qi接jie近jin自zi發fa性xing的de沈shen澱dian狀zhuang態tai時shi,蛋dan白bai質zhi分fen子zi將jiang在zai整zheng齊qi的de堆dui棧zhan下xia形xing成cheng晶jing體ti。舉ju例li來lai說shuo,我wo們men將jiang蛋dan白bai質zhi溶rong於yu低di濃nong度du(~1.0M)的硫酸銨溶液中,將它放置於一密閉含有高濃度(~2.0M)硫酸銨溶液的容器中,由氣相平衡,可以緩慢提高蛋白質溶液中硫酸銨的濃度,進而達成結晶的目的。
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