Recombinant TTR105 115, which does not contain any non-native residues, was prepared as part of a fusion protein construct with a highly soluble B1 immunoglobulin binding domain of protein G (GB1), with typical yields of ∼4 mg/L of uniformly 13C,15N-enriched 13C,15N-enrichedptide per liter of minimal media culture.
A significant advantage of LOW-BASHD detection is that it can be directly incorporated into existing correlation methods, as illustrated here for 2D CACO, NCO, and NCA correlation spectroscopy applied to the β1 immunoglobulin binding domain of protein G and 3D CBCACO correlation spectroscopy applied to the α-subunit of tryptophan synthase.
For GAL1 driven constructs, PATs coding sequences were amplified using oligos that delete the stop codon and cloned in pJV95 vector as fusions to the IgG binding domain of protein A. bsd2Δ tul1Δ strain has been described previously [41].
Figure 2E shows the expression levels of GAL1-driven PATs, tagged at the C-terminus with the IgG binding domain of Protein A. The functionality of all these constructs was tested in complementation assays of the corresponding deletion mutants: Figure 1 for Pfa3, Figure 2 for Swf1, and Figure 3 for Pfa4.
As the bait protein, we used MDM27 300 fused to the TAP tag [25], which contains the IgG binding domain of protein A (ZZ domain), a TEV protease cleavage site and a calmodulin binding peptide for immobilization on IgG beads and specific elution of the MDM2-binding peptide from beads during the affinity selection.
PIP2 breakdown is detected quantitatively in living cells by imaging the translocation of a pleckstrin homology (PH) domain of PLCδ tagged with a fluorescent protein, mCherry (mCh) [9] or a DAG binding domain of protein kinase C β (DBD) tagged with yellow fluorescent protein (YFP) [10], [11], [12].
The pCMV-CBPzz vector contains a SP6 promoter, a part of the omega sequence named O' [40], an N-terminal T7-tag coding sequence, and a C-terminal affinity tag, the coding sequence for the IgG binding domain of protein A (ZZ domain), a TEV protease cleavage site and a calmodulin binding peptide [25].
As structural disordered amino acids in the binding domain of protein may affect the ligand binding with target protein and induce side effect [ 46, 47], the disordered amino acids of PXR protein were predicted before virtual screening.
In the partially purified extracts the antibodies detected bands of about 101, 98 and 108 kD, which correspond to the size expected for the fusion proteins without the 2xIgG binding domain of protein A: AtCuAO1-MYC9-His6, AtCuAO3-MYC9-His6 and AtCuAO3-MYC9-His6, respectively.
