Figure 2

Strategy for the cloning of fusion proteins. Cloning is based on the Multi-site Gateway kit from Invitrogen. The plasmid encoding the ORF of interest can either be an existing entry clone from a Gateway-based ORFeome library, or can be constructed by Gateway-cloning the PCR-amplified ORF into pDONR221 with BP clonase (panel 1). A collection of entry clones encoding the functional modules to be attached to the ORF of interest is constructed similarly by Gateway cloning the PCR-amplified modules into the pDONR P4-P1R (N-term modules) and pDONR P2RP3 (C-term modules) plasmids from the MultiSite Gateway kit (panel 2, see main text for details). All of the inserts in the entry vectors are flanked by appropriate att sites provided by the PCR-amplification primers, and are indicated throughout this figure with a key (e.g. the attB1 site is indicated as B1). Single-fragment Gateway destination expression vectors designed for expression in different model systems are adapted by engineering of the Gateway cassette to allow MultiSite Gateway cloning reactions (panel 3). Such reactions involve one adapted destination vector, as well as the plasmids encoding the ORF of interest and the chosen N-term and C-term modules (white background), whose matching att sites (R4xL4, R1xL1, L2xR2, L3xR3) recombine to produce a destination expression vector encoding the fusion protein in the form of a chimeric cDNA (panel 4). Expression of the cDNA results in the production of a fusion protein whose three constituent parts are linked by short peptides resulting from the translation of the attB1 and attB2 sites remaining after the LR recombination (panel 4, see main text for details).