A COMPARISON OF STRATEGIES TO STABILIZE IMMUNOGLOBULIN FV-FRAGMENTS
A COMPARISON OF STRATEGIES TO STABILIZE IMMUNOGLOBULIN FV-FRAGMENTS
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DOI：
10.1021/bi00458a002
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发表时间：
1990-02-13
期刊：
影响因子：
2.9
通讯作者：
PLUCKTHUN, A
中科院分区：
文献类型：
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作者：
GLOCKSHUBER, R;MALIA, M;PLUCKTHUN, A
抗体的 Fv 片段可能在低蛋白质浓度下解离，并且对于生理温度下的许多应用来说太不稳定。为了稳定 Fv 片段免于解离，我们测试并比较了在大肠杆菌中表达和分泌的已充分表征的磷酸胆碱结合抗体 McPC603 的 Fv 片段的三种不同策略：可变结构域的化学交联、引入分子间二硫键以及构建肽接头以产生“单链”Fv 片段。所有连接的片段显示的半抗原亲和力几乎与整个抗体的亲和力相同，与蛋白质浓度无关，并且对于不可逆的热变性具有显着（高达 60 倍）的稳定性。所有基因工程连接的 Fv 片段都可以在大肠杆菌中以天然构象获得。所报告的生成具有改善的理化性质的 Fv 片段的策略可能会扩展其在生物技术以及治疗和诊断应用中的用途。
Fv-Fragments of antibodies may dissociate at low protein concentrations and are too unstable for many applications at physiological temperatures. To stabilize Fv-fragments against dissociation, we have tested and compared three different strategies on the Fv-fragment of the well-characterized phosphocholine binding antibody McPC603 expressed and secreted in Escherichia coli: chemical cross-linking of the variable domains, introduction of an intermolecular disulfide bond, and constuction of a peptide linker to produce a "single-chain" Fv-fragment. All the linked fragments show hapten affinities nearly identical with that of the whole antibody independent of protein concentration and are significantly (up to 60-fold) stabilized against irreversible thermal denaturation. All genetically engineered linked Fv-fragments can be obtained in native conformation in E. coli. The reported strategies for generating Fv-fragments with improved physicochemical properties may extend their usefulness in biotechnology as well as therapeutic and diagnostic applications.