|
|
||
|
|
|
K Y L E
A . A N D E R S O N
C U R R
E N T R E S E A R C H / I N T E R E S T S My current research involves the expression and
purification of MAGE-A3, a potential cancer vaccine. MAGE-A3 is one member of a large
family of MAGE proteins that are clustered on the X-chromosome. Expression of many of these proteins
is limited to the testicular and possibly ovarian tissues under normal
conditions, but in many cancerous lesions, many members of the MAGE family
are expressed, particularly MAGE-A3.
Its processing and display on MHC class I molecules by the tumor cells
make it an excellent candidate for vaccination. Current expression efforts are focusing on using Escherichia
coli and Pichia pastoris. Fermentation methods using E. coli are well
established, and I have been able to produce intracellular MAGE-A3 as a
soluble protein. Using this
material, I am currently optimizing a purification strategy using anion
exchange and hydrophobic interaction chromatography. Histidine tagged MAGE-A3 constructs
have also been created and can be expressed by E. coli, which may be
necessary for the efficient purification of MAGE-A3 from the numerous E.
coli proteins that carry through the purification process. Difficulties in purification from E. coli derived
protein highlights the importance of using the P. pastoris expression
system. P. pastoris is a
methylotrophic yeast capable of growth using methanol as its sole carbon
source. The media for
fermentation is very simple and well defined. Our primary goal with all of our vaccine targets is to use
P. pastoris to secrete the protein of interest into the culture
media. Removal of the cells at
the end of fermentation gives a highly enriched protein sample, as P.
pastoris secretes very few native proteins. Expression of various proteins in P. pastoris
in our lab has proven to be very hit-or-miss, with some proteins expressing
to very high yields while others express very little. MAGE-A3 has proven to be one of the
latter. Strategies to limit
protease activity and the use of alternate nitrogen sources are being
explored, and work being done by others in the group concerning proteases
will also be exploited to achieve effective protein secretion. E D U C
A T I O N Rochester Institute of
Technology, Rochester, NY, USA Cornell
University, Ithaca, NY, USA R E C O
M M E N D E D L I N K S · Ludwig Institute for Cancer Research ( http://www.licr.org/ ) C O N T
A C T I N F O Cornell University/Ludwig
Institute for Cancer Research Partnership email: kaa26@cornell.edu |
|
[ Home | Mission | Research | Personnel | Calendar | Research Partners & Facilities | Positions Available | Funding | Contact us ] |
||
© 2003