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PCR Cloning and Subcloning> | … PCR products were fused by overlap extension PCR, and the resulting product was recombined with linearized vector in a 30 min RT CloneEZ reaction (GenScript) and transformed to E. coli. To facilitate subsequent cloning in pSSDSfiIPacI-FLAGV5-Gal1, the small stuffer … | Get A Quote |
While transcriptome- and proteome-wide technologies to assess processes in protein biogenesis are now widely available, we still lack global approaches to assay post-ribosomal biogenesis events, in particular those occurring in the eukaryotic secretory system. We here develop a method, SECRiFY, to simultaneously assess the secretability of >10 protein fragments by two yeast species, S. cerevisiae and P. pastoris, using custom fragment libraries, surface display and a sequencing-based readout. Screening human proteome fragments with a median size of 50-100 amino acids, we generate datasets that enable datamining into protein features underlying secretability, revealing a striking role for intrinsic disorder and ... More