Skip to main content
[ "article:topic", "showtoc:no", "license:ccbyncsa", "authorname:coconnor" ]
Biology LibreTexts

15.3: Plasmid-encoded proteins have C-terminal tags

  • Page ID
    17589
  • In this lab, we will be using antibodies to detect Met and LacZ fusion proteins expressed in transformed S. cerevisiae. These fusion proteins have been engineered to add several functional elements to the C-termini of Met and LacZ proteins. Consequently, the fusion proteins expressed from the pBG1805 (Gelperin et al., 2005) and pYES2.1 plasmids have C-terminal tags that

    can interact with a variety of reagents. Each of the plasmids encodes epitope tag(s) that can be detected with antibodies on western blots. These epitopes correspond to highly immunogenic amino acid sequences on the surfaces of viruses that have been shown to be potent inducers of antibody production. The pBG1805 sequence encodes an 9-amino acid sequence of the human influenza virus hemagglutinin (HA) protein (Sleigh et al., 1981), while the pYES2.1 plasmid encodes a 15-amino acid sequence from the P protein of simian virus V5 (Southern et al., 1991). In our blots, we will use a monoclonal antibody directed against the V5 protein, hereafter referred to as anti-V5, to detect proteins expressed from pYES2.1-based plasmids.

    15-fusionproteins.jpg

    The pYES2.1 plasmid also encodes a tag consisting of 6 histidines. This His6-tag binds tightly to metal ions, so it is commonly used to purify overexpressed proteins by passing them through a resin with immobilized zinc or cobalt ions. Unfortunately the His6-tag is not very immunogenic, so it is rarely used in western blots. Together, the V5 and His6 tags add ~5 kDa to the molecular weight of the expressed protein.