Transcript Document

Research Experience in Molecular Biotechnology & Genomics
Summer 2007
Center for Integrated Animal Genomics
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
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.
DISCUSSION
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.
BACKGROUND
•The protein was stored in a -80 degrees freezer.
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.
ASATOR
GFP
CHROMATOR
GFP
GST
MEGATOR
GFP
SKELETOR
GFP
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.
ACKNOWLEDGEMENTS
Colonies of
Bacteria
Eluting beads
RESULTS
Recombinant
Spindle Proteins
REFERENCES
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.
Plasmid
Media plate
Bacteria Culture
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.
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.
Concentrating
Proteins
•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
Spot Testing
1st Protein Gel
5th Protein Gel
6th Protein Gel
9th Protein Gel
Program supported by the National Science Foundation Research Experience for Undergraduates
DBI-0552371