Engineered Als5p Variants in Yeast

V5-tagged Als5p^WT was generated by restriction digestion of pGK114 with BamHI and XhoI which released the Als5p sequence from the vector backbone [39] (link). This backbone was then ligated to: an oligonucleotide, which contains sequences for the invertase secretion signal, V5 epitope tag, flanked by a 5′-BamHI and 3′-NotI restriction sites [54] (link). The coding region of Als5p between a 5′ NotI site and a 3′XhoI site was generated by PCR and was ligated to the modified vector to make pJL1. The resulting construct was verified by sequencing (GeneWiz, South Plainfield, NJ).
Als5p^V326N was generated by digestion of pGK114 with SphI and AleI to generate a 363bp fragment at nucleotide position 1242 to 1605, containing the target sequence to be mutated. This fragment was subcloned into pGEM-T vector and mutagenized using Quickchange (Agilent Technologies, Santa Clara, CA) with mutagenic primer 5′-GAA TAG TGA TGC CGG ATC TAA CGG TAT TAA CAT TGT TGC TAC AAC TAG AAC AGT TAC AGA CAG-3′. The correct mutation was verified by sequencing. The mutated fragment was released from the vector with the same enzymes used in its generation, and placed into the corresponding position of pJL1. The resulting product, pJL1^V326N was verified by sequencing to determine the presence of the full-length Als5p^V326N.
pJL-EV was produced by restriction digestion of pJL1 with BamHI and XhoI and ligating in the multiple cloning site from p414 (ATCC, Manassas, VA). The pJL plasmids were transformed into S. cerevisiae strain W303-1B.