Purification of the HTT N-HEAT (81-1643) with various buffer conditions

Previously, I purified the HTT N-HEAT_81-1643 domain and showed that the HTT N-HEAT_81-1643 domain elutes early from a Superose 6 column 10/300 (https://zenodo.org/record/3462496#.XjsVXSNOk2w). Initial biophysical characterization by DLS showed samples of the HTT N-HEAT_81-1643 construct are made up of large particles ( https://zenodo.org/record/3562523#.Xjw57iN, https://zenodo.org/record/3561096#.Xjw6ByNOk2w).   Thus, our initial results all pointed out at having high oligomeric states of the HTT N-HEAT_81-1643 construct in solution.

To rule out the possibility of a weak interaction with nucleic acid material which could cause the large particle size in solution, we tested whether we could remove the nucleic acid material by adding an additional purification step with heparin resin.  However, purification using the additional heparin step showed no improvement in the purity of the sample from other protein impurities or nucleic acid material  (https://zenodo.org/record/3562523#.XjsVyyNOk2w).

Determination of ideal buffer conditions for the HTT N-HEAT_81-1643 construct using DSLS and DSF (https://zenodo.org/record/3562523#.Xjw57iN

https://zenodo.org/record/3519364#.Xjw6ICNOk2w, https://zenodo.org/record/3561087#.Xjw6LiNOk2w) was not possible as no significant changes in the T_aag or T_m were observed for the conditions tested. Further, the data for both DSF and DSLS could have presented challenges to fit (e.g. The DSF data showed very high initial fluorescence while the DSLS data showed the T_agg ­ for this construct was at the end of the detection limit ~ 90 °C).

Thus, to further assess if we could find an ideal buffer condition where the HTT N-HEAT_81-1643 construct is the most stable and monomeric, we tested different purification conditions.