High-throughput cloning and expression library creation for functional proteomics

Fernanda Festa, Jason Steel, Xiaofang Bian, Joshua LaBaer

Research output: Contribution to journalArticle

27 Scopus citations

Abstract

The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particularly important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single-gene experiments, creating the need for fast, flexible, and reliable cloning systems. These collections of ORF clones can be coupled with high-throughput proteomics platforms, such as protein microarrays and cell-based assays, to answer biological questions. In this tutorial, we provide the background for DNA cloning, discuss the major high-throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and CreatorTM DNA Cloning System) and compare them side-by-side. We also report an example of high-throughput cloning study and its application in functional proteomics. This tutorial is part of the International Proteomics Tutorial Programme (IPTP12).

Original languageEnglish (US)
Pages (from-to)1381-1399
Number of pages19
JournalProteomics
Volume13
Issue number9
DOIs
StatePublished - May 2013

Keywords

  • Gene libraries
  • High-throughput functional studies
  • ORF libraries gene transfer
  • Protein expression
  • Recombination and restriction enzyme-based cloning
  • Technology

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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