Yeast Two-Hybrid CDNA Library Construction And TMEFF2 Interacting Protein Screening

TMEFF2, which encodes a putative transmembrane protein containing twofollistatin-like domains and one epidermal growth factor(EGF)-like domain, is anovel gene related to nerve development found in recent years. It's function is stillunknown to us and the research as to it returns the less. In previous reseach of ourlaboratory, differentially expressed cDNA library was constructed by suppressionsubtractive hybridization (SSH) between left and right forebrain of left retinadeprived pigeon, and some differentially expressed tags(ESTs) were confirmed byreverse Northern blot. One full length cDNA was cloned from these ESTs throughrapid amplification cDNA ends(RACE). It was proved to be TMEFF2 gene byhomologous comparation. To study its function, EGF-like domain of TMEFF2 genewas cloned into pGBKT7 vector as a 'bait' protein to interact with human braincDNA library, and the interaction clones screened out were confirmed to be Celsr2,G3BP2,agrin and prosaposin by sequence analysis. Therefore it was primarilyconcluded that TMEFF2 gene may relate to nerve development and signaltransduction.For the function and mechanism research, the short cytoplasmic region of TMEFF2was cloned into pGBKT7 vector firstly. After the test of DNA-BD/bait protein fortranscriptional activation, toxicity effects and target protein expression, TMEFF2gene was confirmed to be fit for the yeast two hybrid. Secondly, with high frequencyhomologous recombination phenominon in vivo, the yeast two hybrid cDNA libraryof human brain was constructed by CLONTECH SMART methods. The first chain ofcDNA was synthesized by reverse transcription from human brain total RNA and thedouble strands cDNA was amplified by long distance-PCR. Finally, the cDNA librarywe constructed was screened after mating between yeast AH109 and Y187 on thebase of CLONTECH MATCHMAKER SYSTEM 3. Eventually, two putativepositive clones was obtained. One of them is human ryanodine receptor 2 (RYR2)andthe other is an unknown gene-cDNA DKFZp686C06106. As a result, the potentialG-protein activating motif in cytoplasmic region of TMEFF2 may play a role in the signal transduction mediated by Ca²⁺ or tyrosine protein kinase(TPK).Unexpectedly, a new splicing variant sequence of TMEFF2 was obtained when full lengthcDNA of TMEFF2 was cloned. It was showed that the absent 60bp sequence just lies in theseventh exon by sequence analysis. It is to be confirmed by further experience about how thesplice variant works and its proportion in different tissues and the distribution frequency in thepopulation.