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Alternative way of binding: Jun/Fos
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| two human c-Fos:c-Jun:DNA complexes |
Introduction
We also thought of an alternative way of binding of the heterodimeric
Fok to the DNA avoiding the necessity of labelled oligos and their
binding proteins using the binding domain of a transcription factor as
a new adapter. We focused on the binding domain of the activator
protein-1 (AP-1) a crucial transcription factor implicated in numerous
cancers. The protein is composed by a series of dimers. Nine homologues
of the AP-1 leucine zipper region have been characterized, among them
c-Fos, c-Jun and semirational library-designed winning peptides FosW
and JunW. Via leucin zipper they interact among each other and with
their basic region they bind DNA.
Methods
Now we ordered plasmids encoding the sequences for the bZip domains of
c-Fos linked to a His-tag for the purification which could then be
linked to Fok_a and expressed as soluble proteins in Escherichia coli.
First we fused Splitli-Fok_a with Fos and will express this hybrid
protein simultanously with GST-FosW. The His_Fos-Splitli-Fok_a,
GST-FosW and His-FluA-Splitli-Fok_i constructs were cloned into our
expression vector pEx under use of the iGEM compatible restriction
sites under the lac control system to avoid any deleterious effect to
the cell with an ampicillin resictance. RV308 and BL21de3 served as the
host for the experiments. After induction of the recombinant cells with
0.6-0.7 mM isopropyl-beta-D-thiogalactopyranoside (IPTG), the hybrid
proteins will be purified to homogeneity by a gluthation column binding
FosW, followed by the purification of Fos-SplitLi-Foka through binding
at FosW. The GST column was loaded with glutathione.
For an analysis of the cleavage activity of the
Fos-Splitli_Fok_a construct together with FosW we will use reporter
plasmids containing the target recognition sequences of Fos and FosW as
substrates. There are two classes of core sequences, the sequences TRE
(TGACTCA) and CRE (TGACGTCA), which were recognized by the different
AP-1 factors. In a mixture of the expressed protein Fos-Splitli-Fok_a
with GST-FosW, His-FluA-Splitli-Fok_i serving as an expressed Fok_i
protein and the target DNA, Fos should dimerize with FosW. After
activation of the Fok_i and Fok_a partners, the DNA should be cut.
Results and Discussion
The cloning of the construct His_Fos and Splitli-Foka into the pEX
expression vector was done by a triple ligation. The first insert,
His_Fos was cut out by XbaI and AgeI, the second insert, Splitli-Fok_a
by NgoMIV and PstI, the vector was cut open with XbaI and PstI. The
cloning was successful and the concentration of the plasmid and the
sequencing of clones were good.
| Sample ID | User ID | Date | Time | ng/ul | A260 | A280 | 260/280 | 260/230 | Constant | Cursor Pos. | Cursor abs. | 340 raw |
| pExFosSplitFokaK1 | FreiGEM | 14.10.2009 | 19:36 | 76.19 | 1.524 | 0.758 | 2.01 | 2.16 | 50.00 | 230 | 0.705 | -0.009 |
| pExFosSplitFokaK2 | FreiGEM | 14.10.2009 | 19:37 | 50.13 | 1.003 | 0.491 | 2.04 | 2.21 | 50.00 | 230 | 0.453 | 0.078 |
The expression of the single parts Fos-Splitli-Fok_a, GST-FosW and His-FluA-Splitli-Fok_i was done. His-FluA-Splitli-Fok_i was also successfully purified via a Ni-NTA column. The western blot shows bands of the expected size of 46,7kDa in the pooled elution fraction. Purification and in vitro cutting assays will follow.
As a prospect the additional feature of photo-switchability of
Fos
can be generated, allowing regulation of the enzyme’s
activity.
Introduction of cysteine residues as reactive sites into Fos and fusion
of thiol-reactive cross-linkers to them results in intramolecular
cross-linking of the Fos protein. By a photo-switch between cis and
trans conformations these cross-linkers can change their end to end
distance. Thus isomerization can be used to promote helix folding or
unfolding of Fos.
