Cloning, Expression and Purification of HisSumoMet18TevStrep, and Chemical Crosslinking of Proteins in
the targeting Complex of Cytosolic Iron Sulfur Cluster Pathway
Zanub Hassan, Amanda Vo, and Deborah Perlstein
Department of Chemistry, Boston University, Boston MA 02215
ABSTRACT
In yeast, the 4Fe-4S cluster needed by
proteins located in the nucleus and in the
cytosol are assembled and inserted by
the Cytosolic Iron Sulfur Cluster Assembly
(CIA) pathway. In the cytosol, there is a
three protein complex called the CIA
targeting complex that identifies apotargets (without 4Fe-4S) in order to
assemble them to their cofactor. This
targeting complex comprised of the
protein Cia 1, Cia 2, and met18 which are
yeast proteins. However, the mechanism
by which this occurs has not been
established. One thing that has limited
our ability to understand apo-enzyme
recognitions has been problems that has
to do with purification of the individual
components of the CIA targeting complex
BACKGROUND
In the cytosol, there is a three protein
complex called the CIA targeting complex
that identifies apo-targets in order to
assemble them to their cofactor. This
targeting complex comprised of the
protein Cia 1, Cia 2, and met18 which are
yeast proteins.
Construct
Expression and Purification
Elutions
120kDa
120kDa
Our StrepMet18 protein expressed at 120kDa and
we purified it using a strep affinity column.
The top construct is what we have, and the one below is
what we want.by primers we designed
DSS Crosslinking
Cloning by Q5 method
The incubation of Cia 1 and Cia 2 formed a
crosslinked product.
CONCLUSIONS
By optimizing annealing temperature, and concentration,
HiSumoMet18TevStrep amplified at a length of 7kbp.
. With all the components of the targeting complex at
our disposal, interaction testing within complex, and
with apo-proteins can be done
. The DSS crosslinker can be used to stabilize dynamic
interactions that exists within the complex, to enable us
do a biochemical characterization of the complex.
REFERENCES
Amanda Vo
Deborah Perlstein