We established a high-yield purification protocol for recombinant prokaryotic expression of wild-type and variant MCAD proteins using a maltose binding protein (MBP -tag.
Next, a portable Crm1p-binding NES from HIV-1 Rev protein that functions in yeast was fused en masse to the C-terminus of variant Upf3 proteins using loxP sites recognized by bacterial cre-recombinase.
To examine the effect of Mn2+ as a prosthetic group (instead of Mg2+) on normal and translesion polymerase activities by six variants, standing-start primer extension experiments were done with the wild-type and variant pol ι proteins using 24-mer primers annealed to 36-mer templates containing G, N-EtG, O-MeG, 8-oxoG, or an abasic site in the presence of all four dNTPs and 0.15 mM MnCl2.
However, these assays require expression and purification of recombinant MMR proteins (Nystrom-Lahti et al, 2002; Heinen et al, 2002), and although MMR repair in vitro may be deemed normal when sufficient quantities of the variant protein is used, some variants are poorly expressed, suggesting that the cellular levels of the variant may be lower (Kariola et al, 2002).
Intracellular trafficking of AQP2 variant proteins was studied using transient expression of mutant dynamin2-K44A-GFP protein and AQP2 variant protein phosphorylation levels were assessed by Western blotting analysis.
Significances for the differences in T1/2 and t1/2 between wild-type and variant proteins were calculated using one-way ANOVA followed by a Dunnett's post test.
In order to determine whether these mutations lead to an alteration in the ATPase activity, we subjected the purified PD variant proteins to ATP hydrolysis using a well-established single-turnover ATPase assay.
This result also demonstrated that the engineered RecA variant (RecA-Htg) protein used in our experiments was capable of promoting the swarming motility process.
For example, the cancer-restricted splice variant protein could be used as the target for specific antibodies conjugated to tumor cell toxins for cancer treatments.
Wildtype and variant proteins of BMPR-IA were purified using an identical protocol, thus differences in the purification could possibly indicate BMPR-IA variants with non-native fold.
