Screening of human pancreatic cDNA expression library for interacting sequences with normal and oncogenic Rasp21 using yeast two-hybrid analysis

Ras stands for rat sarcoma, which is a family of oncogenes involved in many human cancers. There are three mammalian Ras genes, H-Ras, K-Ras and N-Ras, whose protein products play a critical role in signal transduction, and hence in normal cell growth and division. In this study, a yeast two-hybrid system approach was used to elucidate an interactome within pancreatic cell using the 189 amino acids coding sequences for normal H-Ras and mutant H-Ras. The Ras cDNAs were subcloned into the pGBKT7 bait vector for yeast two-hybrid analysis using PCR. During the study, an inexpensive and very rapid PCR method was developed to directly screen the recombinant clones without further subculturing. The pGBKT7 subclones were validated for the presence of Ras through dideoxy sequencing. Sequential transformations were used to initially transfer the pGBKT7 plasmid containing Ras cDNA insert and subsequently the pGADT7 plasmid containing the pancreatic cDNA library, into yeast strain Y187. The presence of both the plasmids (pGBKT7 and pGADT7) in the Y187 yeast cells was confirmed on---leu/-trp plates. Transformation frequencies varied from 8 to 10 x 104/ug of plasmid DNA. The presence of Ras interacting proteins (including raf) was confirmed on---leu/-trp/-ade/-his plates. For mutant H-Ras, approximately 200 clones were obtained whereas for wild-type H-Ras only two clones were obtained. To identify putative Ras-binding proteins, the pGADT7yeast plasmid DNA (ampicillin resistant) need to be isolated and dideoxy sequenced to identify the cDNA inserts. Once sequenced, the resulting Ras interacting sequence needs to be compared with the known human genome sequence.