Protocol

High-Throughput Cloning of Open Reading Frames (ORFs): Assembling Large Sets of Expression Constructs

¹Vanderbilt University School of Medicine, Nashville, TN 37232-8575, USA
²Harvard University School of Medicine, Harvard Institute of Proteomics, Cambridge, MA 02141-2023, USA

³Corresponding author (joshua_labaer{at}hms.harvard.edu)

INTRODUCTION

Nearly all methods for studying protein function begin with the expression of protein from cloned copies of the protein-coding sequences. Thus, obtaining a complete set of validated protein-coding clones is the first step toward establishing a functional proteomics platform for any organism of interest. Different protein functional studies demand different protein expression vectors; therefore, a flexible vector system should be used that enables the cloned sequences to be transferred rapidly to any vector. This can be achieved most efficiently by using vectors that employ recombinational cloning, a strategy that allows DNA fragments flanked by site-specific recombination sites to be moved from one vector to another in a single-step procedure, in frame and without mutation. There is now extensive experience with several such systems, including the Gateway system (Invitrogen) described here. These reactions are simple enough to allow high-throughput (HT) automation and are highly efficient. Once a "master" clone is created, the identical sequences can be transferred easily into all bacterial, mammalian, and viral vectors commonly used for protein analysis in vivo or in vitro. This protocol describes the process of transferring protein-coding sequences from a master vector into a protein expression (destination) vector for use in functional experiments.