A screening system for identifying interacting proteins using biomolecular fluorescence complementation and transposon gene trap

We have established a new screening system for identifying interacting proteins by combining biomolecular fluorescence complementation (BiFC) and a transposon gene trap system. This system requires creation of a bait strain that stably expresses a fusion product of part of the fluorescent monomeric Kusabira-Green (mKG) protein to a protein of interest. A PiggyBac transposon vector is then introduced into this strain, and a sequence encoding the remainder of mKG is inserted into the genome and fused randomly with endogenous genes. The binding partner can be identified by isolating cells that fluoresce when BiFC occurs. Using this system, we screened for interactors of p65 (also known as RELA), an NF-kappa B subunit, and isolated a number of mKG-positive clones. 5 '- or 3 ' -RACE to produce cDNAs encoding mKG-fragment fusion genes and subsequent reconstitution assay identified PKM, HSP90AB1, ANXA2, HSPA8, and CACYBP as p65 interactors. All of these, with the exception of CACYBP, are known regulators of NF-kappa B. Immunoprecipitation assay confirmed endogenously expressed CACYBP and p65 formed a complex. A reporter assay revealed that CACYBP enhanced 3 kappa B reporter activation under TNF alpha stimulation. This screening system therefore represents a valuable method for identifying interacting factors that have not been identified by other methods.

Automated summary

A new screening system combining BiFC and a transposon gene trap system was established for identifying interacting proteins. This system successfully identified several interactors of the NF-kappa B subunit p65, including PKM, HSP90AB1, ANXA2, HSPA8, and CACYBP, with CACYBP enhancing NF-kappa B reporter activation under TNF alpha stimulation. This screening system provides a valuable method for identifying interacting factors not identified by other methods.