Fusion Constructs (E6901)

  1. Analysis of plasmid DNA: Restriction digests with the same restriction enzymes that were used for cloning the target gene fragment can be used to screen for correct clones, except when SapI is used because the SapI site is lost after ligation. It is also possible to use other restriction sites present in the plasmid.

    Colony PCR or colony hybridization can be used to screen a large number of transformants for the presence of the target gene. See Application Note for "Colony PCR".

  2. Analysis of protein expression: Transform the correct plasmid construct into competent ER2566 or T7 Express. Inoculate 5–10 freshly grown colonies, each in 4 ml LB+Amp media. Grow the culture at 37°C until it reaches an OD600 of ~0.5 or slightly turbid. Transfer 2 ml to a sterile tube as an uninduced control.

    Induce protein expression with 0.4 mM IPTG at 37°C for 2–3 hours or at 15°C overnight. Mix 40 μl culture with 20 μl 3X SDS-PAGE Sample Buffer. Boil for 5 minutes and load 15 μl of both uninduced and induced samples on SDS-PAGE and stain with Coomassie blue.

    Immunodetection with Mouse Anti-CBD Monoclonal Antibody can be used to detect the intein-CBD fusion proteins in total cell lysates. Immunodetection is not necessary if an induced band can be easily visualized by Coomassie blue staining of a SDS-PAGE.

  3. Sequencing: Clones should be further confirmed by DNA sequencing before proceeding to the cell culture and protein expression steps.

  4. Storage: The plasmid encoding the correct fusion protein should be stored at –20°C or a glycerol stock should be made of the cells containing the expression plasmid and stored at –80°C.