Protein structure and analysis single-chain Fv antibody covalently linked to antigen peptides and its structural evaluation.
Protein structure and analysis single-chain Fv antibody covalently linked to antigen peptides and its structural evaluation.
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蛋白质结构与分析 与抗原肽共价连接的单链 Fv 抗体及其结构评价。
DOI：
10.1016/j.ab.2021.114312
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发表时间：
2021
期刊：
影响因子：
2.9
通讯作者：
Oda M
中科院分区：
文献类型：
--
作者：
Hayashi T;Kawasaki M;Kamatari YO;Oda M
单克隆抗体G2特异性识别不同的肽。G2单链抗体通过柔性接头与抗原肽Pep 18 mer和Pep 395共价连接，在大肠杆菌中表达于不溶性部分中，并用盐酸胍溶解，然后复性。我们分析了折叠热力学的复性蛋白质，纯化单体使用尺寸排阻色谱法（SEC）。差示扫描量热法（DSC）结果表明，在有或无抗原肽存在的情况下，抗原肽连接的G2 scFv的热稳定性均高于无抗原的G2 scFv。折叠热力学进一步说明了抗原-抗体亲和力对分子内相互作用的影响。SEC和DSC实验的结合可以证实抗原肽连接的G2 scFv的折叠正确性，并且可以在难以应用抗原结合等“功能筛选”的情况下应用于重折叠蛋白的“结构筛选”。本发明的共价连接肽的方法将有助于稳定的复合物结构，并且将被广泛应用于识别肽抗原的其他抗体。
The monoclonal antibody G2 specifically recognizes different peptides. The single-chain Fv (scFv) antibodies of G2 covalently linked to antigen peptides, Pep18mer and Pep395, via a flexible linker were expressed inEscherichia coliin the insoluble fraction, and were solubilized using guanidine HCl, followed by refolding. We analyzed the folding thermodynamics of the refolded proteins, purified as monomers using size-exclusion chromatography (SEC). The results of the differential scanning calorimetry (DSC) showed that the thermal stabilities of antigen peptide-linked G2 scFvs were higher than those of antigen-free G2 scFv in the absence or presence of antigen peptides. The folding thermodynamics further indicated how the antigen-antibody affinity affect the intramolecular interactions. The combination of SEC and DSC experiments could confirm the folding correctness of antigen peptide-linked G2 scFvs and could be applied for “structural screening” of refolded proteins in the case that the “functional screening” like antigen binding is difficult to apply. The present method to covalently link the peptide would contribute to the stable complex structure, and would be widely applied to other antibodies recognizing peptide antigens.