Proba K, Wörn A, Honegger A, Plückthun A
Biochemisches Institut, Universität Zürich, Switzerland.
J Mol Biol. 1998 Jan 16;275(2):245-53. doi: 10.1006/jmbi.1997.1457.
We generated stable and functional cysteine-free antibody single-chain fragments (scFv) lacking the conserved disulfide bonds in both VH and VL. This was achieved by molecular evolution, starting from the scFv fragment of the levan binding antibody ABPC48, which is naturally missing one of the conserved cysteine residues, by using DNA shuffling and phage display. Several of the selected sequences were expressed and the resulting scFv proteins characterized by equilibrium urea denaturation. Three of the characterized proteins exhibit thermodynamic stability similar to the wild-type protein, and these cysteine-free mutant proteins can now be expressed in functional form in the Escherichia coli cytoplasm. We believe that such molecules are of great utility for use as intrabodies, can be produced by simpler expression strategies and may give further insight into the folding and stability of the immunoglobulin fold.
我们生成了稳定且具有功能的无半胱氨酸抗体单链片段（scFv），其VH和VL中均缺乏保守的二硫键。这是通过分子进化实现的，从天然缺少一个保守半胱氨酸残基的果聚糖结合抗体ABPC48的scFv片段开始，利用DNA改组和噬菌体展示技术。对几个选定的序列进行了表达，并通过平衡尿素变性对所得的scFv蛋白进行了表征。其中三个表征的蛋白表现出与野生型蛋白相似的热力学稳定性，并且这些无半胱氨酸的突变蛋白现在可以在大肠杆菌细胞质中以功能形式表达。我们认为，此类分子作为胞内抗体具有很大的用途，可以通过更简单的表达策略进行生产，并且可能会进一步深入了解免疫球蛋白折叠的折叠和稳定性。
