This document summarizes Hannah Montague's work to purify and conjugate a modified aequorin protein (C5Y82F aequorin) to an anti-CD33 antibody for potential use in detecting acute myeloid leukemia cells. Key steps included adding a histidine tag to C5Y82F aequorin to enable its purification, expressing the tagged protein in E. coli, purifying it using nickel affinity chromatography, and conjugating it to an anti-CD33 antibody, though optimization of conjugation conditions is still needed. The ultimate goal is to use the bioluminescent property of aequorin to detect CD33-expressing leukemia cells.

1. Hannah Montague
Pine Crest School
University of Miami Medical Campus

2. Background
Aequorin- A bioluminescent protein, isolated from the jellyfish Aequrea victoria
and commonly utilized tool to localize and quantify the expression of a variety of intracellular molecules.
Aequorin
Ni/NTA Purification- Common method Protein
utilized to purify aequorin from contaminant proteins that
may alter its kinetic properties. Works through the use of Nickel
and a 6-Histidine tag present on the protein of interest.
C5Y82F Aequorin- A synthetically altered Coelenterazine
mutant of aequorin that does not carry the 6-Histidine tag Molecule
necessary for purification, but includes useful mutations that both increase aequorin’s bioluminescent
half life (tyrosine to phenylalanine) and allow for the attachment of various antibodies (threonine to
cysteine)

3. Purpose
Aequorin protein
1. Purification of C5Y82F aequorin
-In order to enable the purification of C5Y82F aequorin through the
use of the Ni/NTA purification system, a 6-Histidine tag must be added to
the C5Y82F aequroin mutant through the use of a vector
2. Conjugation of C5Y82F aequroin
-Once successfully purified, aequorin will be conjugated to the
antibody, Anti-Human Siglec-3/CD33

4. Materials and Methods
1. Purification of C5Y82F aequorin
a. Preparation of Escherichia coli cultures containing the protein, C5Y82F
Aequorincultures and isolation of aequorin DNA
b. Annealing of peT 30-Xa/LIC Vector to DNA plasmid (this will give the C5Y82F
aequroin the necessary purification handle to allow Ni/NTA purification, a 6-Histidine tag)
c. Primed Aequorin samples were transformed into BL21(DE3)LysS cells to enhance
protein expression
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.

5. Materials and Methods
D. Protein Purification
-Transformed BL21(DE3)LysS cells lysed through French Press
-Lysate (product of lysed bacterial cells) incubated with Ni/NTA
Agarose beads to allow Nickel to bind to 6-Histidine tag on C5Y82F
aequorin protein
-Product loaded into column- washed and eluted with lysis buffer and
imidazole
-Dialysis performed to rid now pure C5Y82F aequorin protein of
imidazole
Column utilized to perform purification

6. Materials and Methods Cont.
2. Conjugation of Anti-CD33 Antibody to
Aequorin
-Preparation of antibody for conjugation: 100ul of anti-CD33 antibody added to 25ul
of 20mM Sulfo-LC-SPDP (cross linker) reagent in order to create an antibody: cross linker ratio of
1:600.
-Aequorin incubated with DTT (Dithiothreitol) in order to ensure reduction of all Thiol
groups
-Aequroin+DTT and Antibody+Sulfo-LC-SPDP centrifuged in
Zeba Spin Desalting Column
-Resulting mixture filtered in order to remove all unwanted and
unreacted products- produces filtrate (unwanted, unreacted protein and
antibodies) and retentate (aequorin+antibody conjugates)
Zeba Spin Desalting Columns

7. 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.

8. Results- Conjugation
-Filtrate and retentate are both incubated with imidazopyrazine coelenterazine
(one of the components, along with Ca2+, that enables the bioluminscence of
aequorin)
-Luminescent activity of both the filtrate and retentate tested with
OPTOCOMP Luminometer
-Filtrate (unreacted aequorin)= 304025 RLU (relative light units)
-Retentate (successful aequorin+antibody conjugates)= 1262 RLU
-Because desired RLUs are in the 1,000,000’s, both products are incubated
overnight in an attempt to increase activity
-Filtrate after overnight incubation= 13,581,502 RLU
-Retentate after overnight incubation= 37,695 RLU
-SDS-PAGE gel run for both samples produces no bands for filtrate or retentate

9. Discussion
- More active aequorin was present in the filtrate than in the
retentate, signifying that the majority of the aequorin
utilized was not successfully conjugated
- Although there were no bands present for either the filtrate
or the retentate in the SDS-PAGE gel, this most likely
occurred due to the fact that the concentration of either
sample was not large enough to be visible on the gel
- While a longer incubation period does, in fact, increase the
activity of aequorin, it does not help to increase conjugation
yield
- In order to determine the optimal conditions for an
aequroin+antibody conjugation reaction, an optimization
study will be run by altering both the time and temperature
of the reaction

10. Future Research
Acute Myeloid Leukemia cells have CD33 receptors (blue dots in Figure 1) on
the outside. The antibody conjugated to C5Y82F aequorin in the study, anti-
human Siglec 3/CD33 (gray Y shape in Figure 1), is raised against, and is
therefore able to bind to these receptors. The future aim of this study is to
therefore utilize these aequorin-antibody conjugates and the
bioluminescent property of aequorin for the detection of acute myeloid
leukemia cells in populations of healthy cells.
Figure 1
Schematic courtesy of Daniel Scott and Sylvia Daunert

11. 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.
Scott D, Daunert S (2013). Developing Sensors for Diagnostics and
Monitoring of Clincially Relevant Samples. IGRT
Yikrazuul (24 July 2008). Aequorin Ribbon Diagram. Wikipedia
Thermo Scientific.