Lawrence Berkeley National Laboratory

Many proteomics initiatives require the production of large collections of expression clones. While traditional methods of cloning, such as restriction enzyme-based cloning, have been well established on a small scale, they are not adaptable for a high-throughput environment. Methods that facilitate cloning in a high-throughput manner are vital to the success of these initiatives. This chapter describes ligation-independent cloning (LIC), an ideal cloning strategy for high-throughput proteomics. In this system, linear plasmid vector and insert DNA are treated to generate complementary single-stranded overhangs that anneal during a short incubation. In addition to the ease of LIC due to the speed and efficiency of the annealing reaction, other attributes make it an optimal system for cloning large numbers of cDNAs. Since restriction enzymes are not used, insert sequences do not need to be screened for internal sites or modified prior to cloning. Any vector can be made LIC compatible, allowing for the production of proteins with different affinity tags, often using the same insert PCR product. The additional amino acids added to the encoded protein by the LIC sequences are minimal and can be modified if necessary, which is often important when N- or C-terminal extensions affect protein function or stability.