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        Selection of Non-aggregating VH Binders from Synthetic VH Phage-Display Libraries

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        The particular interest in VH antibody fragments stems from the fact that they can rival their “naturally occurring” single-domain antibody (sdAb) counterparts (camelid VH Hs and shark VNAR s) with regard to such desirable characteristics as stability, solubility, expression, and ability to penetrate cryptic epitopes and outperform them in terms of less immunogenicity, a much valued property in human immunotherapy applications. However, human VH s are typically prone to aggregation. Various approaches for developing non-aggregating human VH s with binding specificities have relied on a combination of recombinant DNA technology and phage-display technology. VH gene libraries are constructed synthetically by randomizing the CDRs of a single VH scaffold fused to a gene encoding a phage coat protein. Recombinant phage expressing the resulting VH libraries in fusion with the pIII protein is propagated in Escherichia coli. Monoclonal phage displaying VH s with specificities for target antigens are isolated from the libraries by a process called panning. The exertion of stability pressure in addition to binding pressure during panning ensures that the isolated VH binders are also non-aggregating. The genes encoding the desired VH s selected from the libraries are packaged within the phage particles, linking genotype and phenotype, hence making possible the identification of the selected VH s through identifying its physically linked genotype. Here, we describe the application of recombinant DNA and phage-display technologies for the construction of a phage-displayed human VH library, the panning of the library against a protein, and the expression, purification, and characterization of non-aggregating VH s isolated by panning.
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