Design and Construction of a Designed Ankyrin Repeat Protein (DARPin) Display Library

Curr Protoc. 2024 Jan;4(1):e960. doi: 10.1002/cpz1.960.

Abstract

Protein display systems are powerful techniques used to identify protein molecules that bind with high affinity to target proteins of interest. The initial challenge in implementing a display system is the construction of a high-diversity naïve library. Here, we describe the methods to generate a designed ankyrin repeat protein (DARPin) display library using degenerate oligonucleotides. Specifically described is the construction of a single DARPin repeat module by overlap extension PCR, concatenation of the module by restriction enzyme digestion and ligation, and incorporation of the concatenated modules into a full-length DARPin sequence in a bacterial cloning or display vector containing the hydrophilic N- and C-terminal capping domains. Protocols for PCR amplification of DARPin sequences to estimate diversity of naïve and enriched libraries via next-generation sequencing are included, as is a simple Linux-based program for analysis of naïve and enriched sequences. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of a single DARPin repeat by overlap extension PCR Basic Protocol 2: Concatenation of DARPin repeats Basic Protocol 3: Ligation of internal repeats into cloning/display vector containing N- and C-terminal capping repeats Basic Protocol 4: Estimation of library size and diversity by next-generation sequencing (NGS) Basic Protocol 5: NGS analysis of naïve and enriched libraries.

Keywords: DARPin; DNA library construction; designed ankyrin repeat protein; next-generation sequencing; protein binder; protein display.

MeSH terms

  • DNA Restriction Enzymes
  • Designed Ankyrin Repeat Proteins*
  • Gastrointestinal Agents*
  • Gene Library
  • High-Throughput Nucleotide Sequencing

Substances

  • Designed Ankyrin Repeat Proteins
  • DNA Restriction Enzymes
  • Gastrointestinal Agents