Optimizing the conjugation of c5 y82f aequorin to the
- 1. Hannah Montague University of Miami Medical Campus
- 2. Background What is Aequorin? Aequorin Protein Reporter Protein Ni/NTA Purification Coelenterazine Molecule
- 3. Purpose Purification of C5Y82F Aequorin -6-Histidine Tag Conjugation of C5Y82F -Anti-Human Siglec-3/CD33
- 4. Materials and Methods 1. Purification -Preparation of Cultures and Isolation of DNA -Annealing of peT 30-Xa/LIC Vector to DNA plasmid -Transformation
- 5. Transformation of plasmid containing pET30-Xa/LIC vector with Aequorin gene into BL21(DE3)LysS cells. Each change in pigmentation on both plates represents a colony of BL21(DE3)LysS cells containing pET30-Xa/LIC vector carrying the Aequorin gene, grown on Kanamycin resistant Luria Broth Agar.
- 6. Materials and Methods Protein Purification -Bacterial Cells lysed through French Press -Lysate Incubated with Ni/NTA Agarose beads -Loaded into column- washed and eluted with lysis buffer and imidazole -Dialysis to rid of imidizole
- 7. Materials and Methods Cont. 2. Conjugation -Preparation of Anti-CD33 Antibody for Conjugation -Antibody-Cross linker ratio: 1:600 -Conjugation of Anti-CD33 Antibody to Aequorin
- 8. Results- Purification Verification of protein purification and Factor Xa cleavage. The bottom bands of lanes 3 and 4 (After Factor Xa and Factor Xa capture) express the 26.8 kDa aequorin protein.
- 9. Results- Conjugation -Filtrate and retentate incubated with imidazopyrazine coelenterazine -Activity tested with OPTOCOMP Luminometer -Unreacted (Filtrate)= 304025 -Conjugate (Retentate)= 1262 -Incubated overnight -Filtrate= 13581502 -Retentate= 37695 -SDS-PAGE -No bands present for retentate or filtrate
- 10. Discussion - More active aequorin remained unconjugated - No bands present - Longer incubation will increase activity, but not concentration - Optimization study to alter both time and temperate of conjugation to determine optimal conditions - Flourophore - Luminescent chemical compound - Allow for precise visualization
- 11. Future Research -CD33 Receptors -Detection of myeloid leukemia cells in populations of healthy cells
- 12. References Shimomura. (1995). A Short Story of Aequorin. Biol. BulI. 189: I-5. Lippincott-Schwartz, Patterson. (2003). Development and Use of Fluorescent Protein Markers in Living Cells. Science, New Series, Vol. 300, No. 5616, pp. 87-91. Scott D, Dikici E, Ensor M, Daunert S. (2011). Biolumniscence and its impact on bioanalysis. Annu Rev Anal Chem (Palo Alto Calif) 4:297-319. Rowe L, Dikici E, Daunert S. (2009). Engineering Bioluminescent Proteins: Expanding their Analytical Potential. Analytical Chemistry. Vol 81, No. 21.