Schiweck W, Skerra A
Abteilung Molekulare Membranbiology, Max-Planck-Institut für Biophysik, Frankfurt am Main, Germany.
Proteins. 1995 Dec;23(4):561-5. doi: 10.1002/prot.340230411.
The synthetic antibody model "M41" was rationally designed with a binding site complementary to chicken egg white cystatin as the prescribed antigen. In order to permit comparison between the computer model and an experimental three-dimensional structure of the artificial protein, its X-ray crystallographic analysis was pursued. For this purpose, M41 was expressed as a recombinant Fab fragment in E. coli by medium cell density fermentation employing the tightly regulated tetracycline promoter. The Fab fragment was efficiently purified via a His-6 tail fused to its heavy chain and immobilized metal affinity chromatography. To raise the chances for the productive formation of crystal packing contacts, three versions of the Fab fragment were generated with differing constant domains. One of these, the variant with murine C kappa and CH1 gamma 1 domains, was successfully crystallized by microseeding in a sitting drop. The orthorhombic crystals exhibited symmetry of the space group P2(1)2(1)2(1) with unit cell dimensions a = 104.7 A, b = 113.9 A, c = 98.8 A and diffracted X-rays to a nominal resolution of 2.5 A.
合成抗体模型“M41”是经过合理设计的，其结合位点与作为指定抗原的鸡卵清白蛋白半胱氨酸蛋白酶抑制剂互补。为了能够比较计算机模型与人工蛋白质的实验三维结构，对其进行了X射线晶体学分析。为此，利用严格调控的四环素启动子，通过中细胞密度发酵在大肠杆菌中表达M41作为重组Fab片段。通过与重链融合的His-6尾和固定化金属亲和色谱法高效纯化Fab片段。为了提高晶体堆积接触有效形成的几率，生成了具有不同恒定结构域的三种Fab片段变体。其中一种，即具有鼠源Cκ和CH1γ1结构域的变体，通过坐滴法微量接种成功结晶。正交晶体呈现空间群P2(1)2(1)2(1)的对称性，晶胞参数a = 104.7 Å，b = 113.9 Å，c = 98.8 Å，X射线衍射至标称分辨率2.5 Å。
