Hershenson S, Helmers N, Desmueles P, Stroud R
J Biol Chem. 1986 Mar 15;261(8):3732-6.
Crystallization is the primary rate-limiting step in protein structure determination. It has been our experience over approximately 10 years that crystals are obtained in about 20% of the proteins attempted and that only about 10% of these crystals are sufficiently well ordered to permit atomic resolution structure analysis. In attempts to overcome this limitation, we have investigated the effect on crystallization of microheterogeneity in a protein regarded as pure by conventional criteria. Creatine kinase was purified from rabbit skeletal muscle and crystallized from methylpentanediol. The protein appeared to be nearly pure judging by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high specific activity. The crystals that were obtained were of poor quality, and an extensive survey of precipitants, crystallization conditions, and additives failed to discover conditions from which usable crystals could be obtained. The enzyme was then subjected to a series of further purification steps. After each purification step, the quality of the crystals obtained under almost identical conditions improved. The final purification step was flat-bed isoelectric focusing. Crystals grown from focused creatine kinase are well ordered and diffract to approximately 3-A resolution.
结晶是蛋白质结构测定中的主要限速步骤。在大约10年的时间里，我们的经验是，在尝试结晶的蛋白质中，约20%能得到晶体，而在这些晶体中，只有约10%的晶体排列足够有序，能够进行原子分辨率的结构分析。为了克服这一限制，我们研究了按照传统标准被视为纯品的蛋白质中的微异质性对结晶的影响。从兔骨骼肌中纯化肌酸激酶，并从甲基戊二醇中结晶。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和高比活性判断，该蛋白质似乎几乎是纯的。得到的晶体质量很差，对沉淀剂、结晶条件和添加剂进行广泛研究后，未能找到能得到可用晶体的条件。然后对该酶进行了一系列进一步的纯化步骤。在每一步纯化之后，在几乎相同的条件下得到的晶体质量都有所提高。最后一步纯化是平板等电聚焦。从聚焦后的肌酸激酶中生长出的晶体排列有序，衍射分辨率约为3埃。
