A rate-limiting and costly step in many proteomics analyses is the cloning of all of the ORFs for an organism into technique-specific vectors. Here, we describe the generation of a Campylobacter jejuni expression clone set using a high-throughput cloning approach based on recombination in E. coli. The approach uses native E. coli recombination functions and requires no in vitro enzymatic steps or special strains. Our results indicate that this approach is an efficient and economical alternative for high-throughput cloning.