High-throughput cloning and expression library creation for functional proteomics


  • This Tutorial is part of the International Proteomics Tutorial Programme (IPTP12). Details can be found at: http://www.proteomicstutorials.org/.

  • Colour Online: See the article online to view Figs. 1–7 in colour.

Correspondence: Dr. Joshua LaBaer, Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, 1001 S. McAllister Avenue, Tempe, AZ 85287–6401

E-mail: joshua.labaer@asu.edu

Fax: 480-965-3051


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).