LIC Duet™ Adaptors provide a means to clone two open reading frames (ORFs) simultaneously into one bacterial Ek/LIC plasmid for coexpression in E. coli. The adaptors anneal directionally to the 3′-end of the first LIC target ORF insert and the start of the second LIC target ORF insert, thereby creating a three-part annealed insert: ORF-1/adaptor/ORF-2. This annealed complex in turn anneals to the LIC vector overhangs. Therefore, the first LIC target ORF is fused to an N-terminal tag encoded by the LIC vector and the second LIC target ORF is fused to an N-terminal tag encoded by the LIC Duet Adaptor. The adaptors are designed to be interchangeable because they all possess a common overhang upstream of the T7lac promoter and a different yet common overhang downstream of the enterokinase cleavage site, or the ATG start codon in the Minimal Adaptor. Given the availability of multiple pET Ek/LIC vectors and LIC Duet Adaptors, numerous combinations of coexpressed fusion proteins are possible. Four LIC Duet Ek Adaptors encode a T7lac promoter/operator, strong ribosome binding site (RBS), ATG start codon, and one of the following N-terminal fusions: T7•Tag®, Trx•Tag™, GST•Tag™, or Nus•Tag™. All of the adaptor-encoded tags are useful for the detection of fusion protein. T7•Tag and GST•Tag facilitate purification and the Nus•Tag, Trx•Tag, and GST•Tag may enhance solubility of their fusion partner. An enterokinase cleavage site follows each adaptor-encoded tag for removal if desired. The LIC Duet™ Minimal Adaptor is designed for expression of a target protein with a minimal fusion and does not include an enterokinase cleavage site. Compatible target ORFs are generated by PCR using primers with specified 5′-nucleotide extensions. The target ORF PCR products are purified to remove dNTPs (and original plasmid if it was used as template) and then treated with T4 DNA Polymerase in the presence of dATP to generate the overhangs compatible with the Ek/LIC vector and LIC Duet Adaptor. Cloning is very efficient because only the desired product is formed by annealing. An annealing reaction consisting of a pET, pCDF, pr pRSF Ek/LIC vector, target ORF 1–LIC insert, LIC Duet Adaptor, and target ORF 2–LIC insert is transformed into competent E. colicells. Covalent bond formation at the vector-insert and adaptor-insert junctions occurs within the cell to yield circular plasmid. The verified construct is ready for expression in bacteria. Coexpression of Three to Six Target Proteins By using a cloning strategy that includes LIC Duet Adaptors in combination with compatible pET, pRSF, and pCDF Ek/LIC vectors, it is possible to coexpress three to six target proteins. | 
