Chioma Ebiringa1, Laurence Woodruff 2 , and Kristen Johanse

1
Research Experience in Molecular Biotechnology
Genomics Summer 2007
Center for Integrated Animal Genomics
Isolation and Purification of GST-GFP Protein for
Screening Anti-GFP Antibody
Chioma Ebiringa1, Laurence Woodruff 2 , and
Kristen Johansen 2 1 Department of Natural
Science, Bowie State University, Bowie, MD,
20715 2 Department of Biochemistry, Biophysics,
and Molecular Biology, Ames, IA 50011
ABSTRACT
GOAL
DISCUSSION
The Green Fluorescent Protein (GFP), which is
found in jellyfish, is used in our lab to study
different proteins in Drosophila that make up a
structure called the spindle matrix. We have
made recombinant proteins using GFP, in order to
observe and study their roles inside the cell
during cell division. My project was to make
anti-GFP antibody, which can recognize and bind
to the GFP that was tagged onto each of these
proteins. We can detect the locations of these
spindle proteins within fixed cells by looking at
the location of the antibody. E.coli cells that
were transformed using a plasmid that codes for
GST-GFP protein, were inoculated and induced to
make GFP protein that have an affinity for
Glutathione Agarose beads. Using the GST
Purification Methods, the GST-GFP protein was
harvested from the cells and bound to Glutathione
beads after they were purified, they were then
eluted from the beads and stored for later use.
This protein was very difficult to elute from the
beads. There were times when the protein left on
the beads was greater than all the elutes
combined. At times there was little to no protein
produced by the bacteria. The fifth culture made
a huge difference in the protein amassed, while
the seventh culture produced no protein. The post
binding sups were re-bound to beads at least
twice each time, but the quantity of protein that
was eluted was very small.
The objective of this research is to make 4mg of
GST-GFP protein that would be used to screen the
antibody that was produced.
MATERIALS AND METHODS

• E.coli cells were transformed with cloned GST-GFP
  plasmid.
• They were cultured overnight. Using IPTG as an
  inducer, the bacteria was induced to make GST-GFP
  proteins.
• The protein was isolated from the cell, bound to
  Glutathione beads, and purified.
• The protein was eluted from the beads by using
  Glutathione Elution Buffers.
• 12 SDS PAGE Gels were ran and analyzed to
  estimate the amount of protein that was
  collected.
• The protein was stored in a -80 degrees freezer.

REFERENCES
GST Gene Fusion System Handbook. (Pharmacia
Biotech) Johansen, K.M and J. Johansen (2007).
Cell and Molecular Biology of the Spindle
Matrix. International Review of Cytology. (In
press). Max Planck Institute for Molecular
Genetics. CURL www.molgen.mpg.de. Watson, D.
James, Michael Gilman, Jan Witkowski, Mark
Zoller. Recombinant DNA, 2nd ed W.H. Freeman
and Company 41 Madison Avenue, New York, NY,
1997 p. 63-75.
BACKGROUND
The spindle matrix is a structure that has been
found in the cell to stabilize and direct
chromosomal segregation during mitosis. This
structure interacts with the microtubule spindle
apparatus, but it is independent of it. It can
exist even when the microtubule spindle has been
disassembled. Asator, Chromator, East, Megator,
and Skeletor are proteins found in Drosophila
that are known to be associated with this
fusiform structure. To conduct our studies on
these proteins, recombinant proteins of four of
these proteins was made by tagging them to GFP.
This GFP protein give off green fluorescent light
wherever the spindle proteins are located in the
cell however, there are times (such as when the
cells have been treated with a fixative) when
this green illumination is quenched in the cell,
making it difficult to find the spindle proteins.
For this reason, we are making an anti-GFP
antibody that would bind to all of the GFP and
allow us to locate them. Clones of GST-GFP
plasmids were used to produce GST-GSP protein in
E.coli cells and this protein was used to produce
GFP antibody in mice. These mice are currently
being screened to select the best responder for
production of monoclonal antibodies. For this
purpose, I am inducing more GST-GFP protein that
would be used in screening the hybridoma lines
that will be made by the Hybridoma Facility.
GST
Plasmid
ACKNOWLEDGEMENTS
Colonies of Bacteria
Media plate
I would like to thank Drs. Kristen and Jorgen
Johansen for giving me the opportunity to work in
their lab this summer. I want to thank my
mentor Laurence Woodruff for working with me
throughout this summer. I would also like to
thank Weili Cai, Xiaomin Bao, Hongying Qi, Yun
Ding, Huai Deng, Dr. Jack Girton and all other
workers in the lab for their support. Thank you
Dr. Max Rothschild, Justin Rice, NSF, and Iowa
State University for allowing me to be a part of
this NSF-REU Program. I would also thank Dr.
George Ude, my mentor at Bowie State University.

Bacteria Culture
Eluting beads
Concentrating Proteins
RESULTS

• At the end of five week 400ug of GST-GFP protein
  was produced.
• Some of it was used for spot testing on the
  antibodies that were produced in mice.
• The fifth inoculation produce approximately
  1.25mg of protein
• The sixth culture produced no protein.
• So far, I have made approximately 2.9mg of
  protein needed for screening GFP antibody

GFP
ASATOR
GFP
CHROMATOR
Recombinant Spindle Proteins
GFP
MEGATOR
SKELETOR
GFP
Spot Testing
5th Protein Gel
1st Protein Gel
6th Protein Gel
9th Protein Gel
Program supported by the National Science
Foundation Research Experience for
Undergraduates DBI-0552371