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.