Engineering of fluorophore-polyprotein construct
Mentor 1
Dr. Ionel Popa
Location
Union Wisconsin Room
Start Date
5-4-2019 1:30 PM
End Date
5-4-2019 3:30 PM
Description
Protein L, consisting of 719 amino acid residues, is isolated from the surface of bacterial species Peptostreptococcus magnus and is known to bind to immunoglobulins through L chain interaction. The objective of this project was to engineer and express a polyprotein construct made from eight domains of protein L in series with a fluorescence domain, EGFP, and determine its possible usefulness as a secondary detection reagent for applications such as Western blots. Starting from the monomeric unit, we applied molecular biology protocols and engineered a poly protein L made from eight repeats. This can be classified into three simple steps: monomer to dimer, dimer to tetramer, and finally tetramer to octamer. Two restriction enzyme digestions were followed to cut open the vector (containing octamer) and digest the fragment. The two were then ligated together. The final product was screened in the lab and also sent for sequencing to double check. We have finally expressed this protein and tested its antibody labeling activity using Western blot technique.
Engineering of fluorophore-polyprotein construct
Union Wisconsin Room
Protein L, consisting of 719 amino acid residues, is isolated from the surface of bacterial species Peptostreptococcus magnus and is known to bind to immunoglobulins through L chain interaction. The objective of this project was to engineer and express a polyprotein construct made from eight domains of protein L in series with a fluorescence domain, EGFP, and determine its possible usefulness as a secondary detection reagent for applications such as Western blots. Starting from the monomeric unit, we applied molecular biology protocols and engineered a poly protein L made from eight repeats. This can be classified into three simple steps: monomer to dimer, dimer to tetramer, and finally tetramer to octamer. Two restriction enzyme digestions were followed to cut open the vector (containing octamer) and digest the fragment. The two were then ligated together. The final product was screened in the lab and also sent for sequencing to double check. We have finally expressed this protein and tested its antibody labeling activity using Western blot technique.
