Selecting for antibody scFv fragments with improved stability using phage display with denaturation under reducing conditions

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• 作者：Brockmann ; Eeva-Christine ; Cooper ; Matthew ; Str??msten ; Nelli ; Vehni??inen ; Markus ; Saviranta ; Petri
• 关键词：scFv ; single-chain Fv antibody fragment ; V_H ; heavy chain of an antibody Fv fragment ; V_L ; light chain of an antibody Fv fragment ; GdmCl ; guanidinium chloride ; TSH ; thyroid-stimulating hormone ; DTT ; dithiotreito
• 刊名：Journal of Immunological Methods
• 出版年：2005
• 出版时间：January, 2005
• 年：2005
• 卷：296
• 期：1-2
• 页码：159-170
• 全文大小：259 K

文摘

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 °C 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^H66Arg or Asn^H52aSer mutations, which are known to stabilize other scFvs. Periplasmic expression level of the mutants was also improved.