(Research Papers) Accelerated Screening of Phage-Display Output with Alkaline Phosphatase Fusions

Zhaozhong      Han; Ece      Karatan; Michael   D   Scholle; John      McCafferty; Brian   K   Kay

Abstract

When using multiple targets and libraries, selection of affinity reagents from phage-displayed libraries is a relatively time-consuming process. Herein, we describe an automation-amenable approach to accelerate the process by using alkaline phosphatase (AP) fusion proteins in place of the phage ELISA screening and subsequent confirmation steps with purified protein. After two or three rounds of affinity selection, the open reading frames that encode the affinity selected molecules (i.e., antibody fragments, engineered scaffold proteins, combinatorial peptides) are amplified from the phage or phagemid DNA molecules by PCR and cloned en masse by a Ligation Independent Cloning (LIC) method into a plasmid encoding a highly active variant of E. coli AP. This time-saving process identifies affinity reagents that work out of context of the phage and that can be used in various downstream enzyme linked binding assays. The utility of this approach was demonstrated by analyzing single-chain antibodies (scFvs), engineered fibronectin type III domains (FN3), and combinatorial peptides that were selected for binding to the Epsin Nterminal Homology (ENTH) domain of epsin 1, the c-Src SH3 domain, and the appendage domain of the gamma subunit of the clathrin adaptor complex, AP-1, respectively.

Keywords: alkaline phosphatase, ligation-independent cloning, enth domain, fn3 domain, gamma subunit of ap-1, sh3 domain

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Combinatorial Chemistry & High Throughput Screening

Title: (Research Papers) Accelerated Screening of Phage-Display Output with Alkaline Phosphatase Fusions

Volume: 7 Issue: 1

Author(s): Zhaozhong Han, Ece Karatan, Michael D Scholle, John McCafferty and Brian K Kay

Affiliation:

Keywords: alkaline phosphatase, ligation-independent cloning, enth domain, fn3 domain, gamma subunit of ap-1, sh3 domain

Abstract: When using multiple targets and libraries, selection of affinity reagents from phage-displayed libraries is a relatively time-consuming process. Herein, we describe an automation-amenable approach to accelerate the process by using alkaline phosphatase (AP) fusion proteins in place of the phage ELISA screening and subsequent confirmation steps with purified protein. After two or three rounds of affinity selection, the open reading frames that encode the affinity selected molecules (i.e., antibody fragments, engineered scaffold proteins, combinatorial peptides) are amplified from the phage or phagemid DNA molecules by PCR and cloned en masse by a Ligation Independent Cloning (LIC) method into a plasmid encoding a highly active variant of E. coli AP. This time-saving process identifies affinity reagents that work out of context of the phage and that can be used in various downstream enzyme linked binding assays. The utility of this approach was demonstrated by analyzing single-chain antibodies (scFvs), engineered fibronectin type III domains (FN3), and combinatorial peptides that were selected for binding to the Epsin Nterminal Homology (ENTH) domain of epsin 1, the c-Src SH3 domain, and the appendage domain of the gamma subunit of the clathrin adaptor complex, AP-1, respectively.

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Han Zhaozhong, Karatan Ece, Scholle D Michael, McCafferty John and Kay K Brian, (Research Papers) Accelerated Screening of Phage-Display Output with Alkaline Phosphatase Fusions, Combinatorial Chemistry & High Throughput Screening 2004; 7 (1) . https://dx.doi.org/10.2174/138620704772884823

DOI https://dx.doi.org/10.2174/138620704772884823	Print ISSN 1386-2073
Publisher Name Bentham Science Publisher	Online ISSN 1875-5402

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