Use of thiophilic adsorption chromatography for the one-step purification of a bacterially produced antibody Fab fragment without the need for an affinity tag
Use of thiophilic adsorption chromatography for the one-step purification of a bacterially produced antibody Fab fragment without the need for an affinity tag
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DOI：
10.1006/prep.1999.1142
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发表时间：
1999-12-01
影响因子：
1.6
通讯作者：
Skerra, A
中科院分区：
文献类型：
--
作者：
Fiedler, M;Skerra, A
用亲硫型吸附层析(TAC)从大肠杆菌周质蛋白组分中分离纯化了重组抗体IN-1的FEB片段。在高浓度的硫酸铵作用下，F-ab片段被吸附到T-凝胶上，并且不依赖于Hiss标签或Strep标签的存在，也不依赖于C(H)1和C-L结构域的人或小鼠的性质(IgG1kappa亚类)。洗脱是通过降低盐梯度来实现的，产生具有正确组装的、高纯度的异源二聚体F-ab片段的级分。有趣的是，F-ab片段的CDR-L1中丙氨酸残基与苯丙氨酸的单一取代显著提高了柱上的保留，因此定量洗脱需要长时间应用低盐缓冲液。我们的发现表明，TAC一般适合于细菌产生的F-ab片段的分离，并支持芳香族侧链在与亲和基质的相互作用中发挥重要作用的观点。这种方法在生产体内应用的蛋白质方面应该被证明是有价值的，就像促进中枢神经系统轴突再生的抗体IN-1的F-ab片段的情况一样，(C)1999 Academy Press。
Thiophilic adsorption chromatography (TAC) was employed for the purification of a recombinant Feb fragment of the antibody IN-1 from the periplasmic protein fraction of Escherichia coli. Adsorption of the F-ab fragment to the T-gel was achieved at a high concentration of ammonium sulfate and turned out to be independent of the presence of a Hiss tag or Strep tag or of the human or murine nature of the C(H)1 and C-L domains (subclass IgG1/kappa). Elution was effected by means of a decreasing salt gradient, yielding fractions with the correctly assembled, heterodimeric F-ab fragment at high purity. Interestingly, the single substitution of an alanine residue with phenylalanine in the CDR-L1 of the F-ab fragment significantly enhanced the retention on the column so that quantitative elution necessitated prolonged application of a low-salt buffer. Our findings suggest that TAC is generally suitable for the isolation of bacterially produced F-ab fragments and support the notion that aromatic side chains play an important role in the interaction with the affinity matrix. This method should prove valuable in the production of proteins for in vivo applications as might be the case for the F-ab fragment of the antibody IN-1, which promotes axonal regeneration in the central nervous system, (C) 1999 Academie Press.