A preliminary study towards the construction of Carboxypeptidase Y - Legumin gene fusions to facilitate an analysis of protein sorting in yeast

Abstract

In an attempt to isolate and clone the promotor and preprosequence of the yeast vacuolar protease carboxypeptidase Y, three, PRC-1-derived, Bsc I - Bam HI restriction fragments were ligated into a vector plasmid (pKS(^+)) and separately transformed into E. coli. Preliminary screening and restriction analysis suggested that one transformant (pCPYF3), contained the desired sequences. However, more detailed restriction analyses and ultimately DNA sequencing of the cloned insert, subsequently showed this not to be so. A reappraisal of the data indicated the likely location of the desired PRCl fragment in a different set of transformants (pCPYF2). Screening of these transformants however, yielded an inconclusive result. Legumin encoding cDNA sequences were successfully cloned into pKS(^+). Exonuclease III/mung bean nuclease deletion of both the PRCl and leg A derived sequences was performed in order to edit both genes in preparation for the construction of a PRCl-leg A gene fusion.