Single-domain antibody fragments with high conformational stability
Single-domain antibody fragments with high conformational stability
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DOI：
10.1110/ps.34602
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发表时间：
2002-03-01
期刊：
影响因子：
8
通讯作者：
Matagne, A
中科院分区：
文献类型：
--
作者：
Dumoulin, M;Conrath, K;Matagne, A
多种技术，包括通过傅里叶变换红外光谱、荧光、圆二色性和表面等离子体共振光谱监测的高压解折叠，已用于研究源自骆驼重链抗体的六种单结构域抗原结合剂的平衡折叠特性。对溶菌酶、β-内酰胺酶和染料（RR 6）具有特异性。各种变性条件（氯化胍、尿素、温度和压力）提供了用于表征抗体片段的稳定性和展开特性的互补和独立的方法。对于所有结合剂，复性后生物活性的完全恢复表明化学诱导的解折叠是完全可逆的。此外，变性实验，随后的光学光谱方法和亲和力测量表明，抗体片段在一个单一的过渡合作展开。因此，解折叠/重折叠平衡通过简单的两态机制（N可逆箭头U）进行，其中仅天然和变性状态被显著填充。然而，热诱导的变性不是完全可逆的，并且结合能力的部分丧失可能至少部分地由于长环（CDR）的不正确重折叠，其负责抗原识别。最有趣的是，所有的片段都对热诱导变性有相当的抗性（表观Tm = 60- 80 ℃），并且显示出高的构象稳定性（Δ G（H2O）= 30-60 kJ mole（-1））。对于任何功能性常规抗体片段，从未报道过这种高热力学稳定性，即使当考虑工程化抗原结合剂时。因此，骆驼科重链抗体片段的减小的尺寸、改善的溶解度和更高的稳定性对于生物技术和医学应用是特别感兴趣的。
A variety of techniques, including high-pressure unfolding monitored by Fourier transform infrared spectroscopy, fluorescence, circular dichroism, and surface plasmon resonance spectroscopy, have been used to investigate the equilibrium folding properties of six single-domain antigen binders derived from camelid heavy-chain antibodies with specificities for lysozymes, beta-lactamases, and a dye (RR6). Various denaturing conditions (guanidinium chloride, urea, temperature, and pressure) provided complementary and independent methods for characterizing the stability and unfolding properties of the antibody fragments. With all binders, complete recovery of the biological activity after renaturation demonstrates that chemical- induced unfolding is fully reversible. Furthermore, denaturation experiments followed by optical spectroscopic methods and affinity measurements indicate that the antibody fragments are unfolded cooperatively in a single transition. Thus, unfolding/refolding equilibrium proceeds via a simple two-state mechanism (Nreversible arrowU), where only the native and the denatured states are significantly populated. Thermally- induced denaturation, however, is not completely reversible, and the partial loss of binding capacity might be due, at least in part, to incorrect refolding of the long loops (CDRs), which are responsible for antigen recognition. Most interestingly, all the fragments are rather resistant to heat-induced denaturation (apparent T-m= 60-80degrees-C), and display high conformational stabilities (DeltaG(H2O) = 30-60 kJ mole(-1)). Such high thermodynamic stability has never been reported for any functional conventional antibody fragment, even when engineered antigen binders are considered. Hence, the reduced size, improved solubility, and higher stability of the camelid heavy-chain antibody fragments are of special interest for biotechnological and medical applications.