High-throughput production of the recombinant proteins expressed in Escherichia coli utilizing cDNA resources

Conventionally, expression plasmids in Escherichia coli have generally been constructed using ligation reaction-assisted cloning followed by the generation of inserts. In such cases, the insert was generated by polymerase chain reaction (PCR), digestion using restriction enzymes, or oligonucleotide synthesis. To overcome the restrictions of these conventional methods, we improved them by utilizing an in vitro site-specific recombination reaction, based on the integrase-excisionase system of bacteriophage lambda to insert DNA fragments. This method enabled us to insert tens of fragments into expression vectors in parallel. We applied these methods to produce glutathione S-transferase (GST)-fused or maltose-binding protein (MBP)-fused proteins in Escherichia coli. As a result, we successfully produced and purified more than 3,000 recombinant proteins for further study of reverse chemical genetics.