Research Paper Volume 4, Issue 6 pp 417—429
DNA binding residues in the RQC domain of Werner protein are critical for its catalytic activities
Figure 2. Purification of the WRN wild type and mutant proteins using new constructs.
(A) The map of 6xHis-WRN-FLAG/pFastBac1-InteinCBDAla construct, total size 9857 bp. (B) Improved WRN purification scheme. (C) SDS-PAGE showing steps of WRN purification. Lane 1, molecular marker; lane 2, whole cell extract; lane 3, pooled fractions from HisTrap column; lane 4, pooled fractions from chitin column. (D) SDS-PAGE of purified WRN variants. Lane 1, molecular marker; lane 2, WRN wild type; lane 3, WRN R993A; lane 4, WRN F1037A; lane 5, WRN R993A/F1037A. (E) Heat denaturation curves of WRN variants. 2 μg of WRN or WRN mutant was incubated with serial dilutions of SYPRO Orange. The curves were obtained by monitoring the change of fluorescence intensity given by qPCR equipment. Observed data was normalized as described in Experimental Procedures.