Tandem Recombineering by SLIC Cloning and Cre-LoxP Fusion to Generate Multigene Expression Constructs for Protein Complex Research

• DOI: 10.1007/978-1-62703-625-2_11
• Publication Date: 2013-08-30T20:48:21Z
• Authors: Matthias Haffke; Cristina Viola; Yan Nie; Imre Berger
• Keywords: Multiprotein Complexes; Genetic Vectors; Gene Expression; Proteins; DNA Shuffling; Cloning, Molecular; Homologous Recombination; Protein Engineering; 3. Good health

A robust protocol to generate recombinant DNA containing multigene expression cassettes by using sequence and ligation independent cloning (SLIC) followed by multiplasmid Cre-LoxP recombination in tandem for multiprotein complex research is described. The protocol includes polymerase chain reaction (PCR) amplification of the desired genes, seamless insertion into the target vector via SLIC, and Cre-LoxP recombination of specific donor and acceptor plasmid molecules, optionally in a robotic setup. This procedure, called tandem recombineering, has been implemented for multiprotein expression in E. coli and mammalian cells, and also for insect cells using a recombinant baculovirus.