Expression of different fragments of integrin beta 3 subunit in the yeast two-hybrid system and their activation of reporter gene

Aim: To construct the bait vectors containing different amino terminal fragments of integnin beta 3 subunit in the yeast two-hybrid system, and to test whether their expressed product could affect the growth of yeast cells and activate the transcription of reporter genes. Methods: The cDNA fragments encoding different sizes of integrin beta 3 amino terminal region were amplified by PCR, then cloned into pAS2-1. These fragments included A1 (55E-199Q), A2(235K-385S), A3 (4 I-454T), A4 (4 I-77S), A5 (4 I-147S) and A6 (4 I-284S). After verified by sequencing, these bait vectors were transferred into the yeast two-hybrid host Y190 to assay whether the transcription of the reporter genes was activated. Results: The bait vectors of different sizes of integnin beta 3 amino terminal region were constructed successfully. It was found that all fragments were not toxic to Y190. But fragment 4 I-77S and another mutant fragment (4 I-F56 + Q) activated the expression of the reporter genes. Conclusion: Fragments A1, A2 and A3 could be used as the bait in the yeast two-hybrid system. The amino terminal fragment (4 I-77S) can activate the transcription of the Gal4-dependent reporter genes when it is fused to the Gal4 DNA binding domain.