Selecting for antibody scFv fragments with improved stability using phage display with denaturation under reducing conditions
Selecting for antibody scFv fragments with improved stability using phage display with denaturation under reducing conditions
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DOI：
10.1016/j.jim.2004.11.008
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发表时间：
2005-01-01
影响因子：
2.2
通讯作者：
Saviranta, P
中科院分区：
文献类型：
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作者：
Brockmann, EC;Cooper, M;Saviranta, P
单链抗体(ScFv)的稳定性可以通过诱变和噬菌体展示选择来提高，其中不稳定的变异体首先通过例如变性处理来灭活。在这里，我们描述了一种改进的策略，用于通过噬菌体展示选择稳定的抗体片段，基于还原条件下的变性。这一策略被应用于抗促甲状腺激素(TSH)单链抗体片段，如果在常规使用的非还原条件下进行变性，则该片段在选择过程中会显着复性。然而，通过结合变性和结构域内二硫键的还原，有效地防止了复性，这为选择稳定性更好的克隆创造了有利条件。利用这一策略，在4-5次浓缩循环后，ScFv突变株的热稳定性和0.8-0.9M的稳定性都得到了提高。选择的最稳定的突变体包含Lys(H)66Arg或Asn(H)52aSer突变，这是已知的稳定其他单链抗体的突变。突变体的周质表达水平也得到了提高。(C)2004爱思唯尔B.V.保留所有权利。
Stability of single-chain Fvs (scFvs) can be improved by mutagenesis followed by phage display selection where the unstable variants are first inactivated by, for example, denaturing treatment. Here we describe a modified strategy for the selection of stabilized antibody fragments by phage display, based on denaturation under reducing conditions. This strategy was applied to an anti-thyroid-stimulating hormone (TSH) scFv fragment which refolded remarkably during the selection if denaturation was carried out in conventionally used non-reducing conditions. Refolding was, however, efficiently prevented by combining denaturation with reduction of the intra-domain disulfide bridges, which created favourable conditions for selection of clones with improved stability. Using this strategy, scFv mutants with 8-9 degreesC improved thermal stability and 0.8-0.9 M improved stability for guanidinium chloride were found after 4-5 enrichment cycles. The most stable mutants selected contained either Lys(H)66Arg or Asn(H)52aSer mutations, which are known to stabilize other scFvs. Periplasmic expression level of the mutants was also improved. (C) 2004 Elsevier B.V. All rights reserved.