Test functionality of the human Trmt1 and Ppic predicted signal peptides by yeast secretion trap (YST)

Secreted and transmembrane proteins are key players in various biological processes. they are accessible to various drug delivery mechanisms and they have a critical role as diagnostic and prognosis factors in clinical states. There are several bioinformatic softwares applied in the prediction of signal peptides and there are several experimental methods to trap signal peptides and identify secreted and transmembrane proteins. YST abbreviated form of yeast secretion trap is one of experimental methods based on pYST vectors and a host Saccharomyces cerevisiae strain 066-2. The host yeast is mutant and lacks invertase. The three pYST expression vectors encoding a mutant invertase in one of three frames and lacking the start codon and signal sequence. In this work we intended to set up YST system and for this purpose we examined two electronically predicted signal sequences in the amino terminal of cyclophilin c (ppic) and tRNA methyl transferase (Trmt1) human proteins. In addition we used putative signal sequence of pi16 as positive control and putative mitochondrial signal sequence of Tfam as the negative control to confirm functionality of the system. these sequences were amplified by PCR and then cloned to in frame in pYST expression vectors. Subsequently, The host yeast strain 066-2 recombinant were transformed with recombinant vectors and cultured on the selective agar media. The results of expriments confirmed the functionally of the system. Neither trmt1 nor ppic amino terminal predicted signal sequence did not function as signal sequence in this system.