Identification of domains in non-core RAG1. (A) MALDI-TOF spectrum of proteolytic products generated during purification of MBP-RAG1(1-380). Two peaks were observed with molecular weight (MWt) values: (1) 52,756.4 Da, consistent with cleavage C-terminal to residue 86, and (2) 44,061.9 Da, consistent with cleavage just C-terminal to the MBP portion of the fusion construct. The predicted mass of MBP-RAG1(1-86) is 52,815 Da. (B) SEC of MBP-RAG1(1-86). (C) SEC of MBP-RAG1(87-380). Vo denotes the void volume and the position at which aggregated protein elutes. Inset: SEC peaks 1-3 analyzed by SDS-PAGE. (All lanes are from the same gel.) Samples in lanes 1-3 correspond to peaks assigned in the SEC elution profile. (D) MALDI-TOF spectrum of peak 2 from the SEC of MBP-RAG1(87-380). Five major peaks were observed with MWt values: (1) 60,902.8, (2) 60,255.1, (3) 58,410.8, (4) 55,239.7, and (5) 43,273 Da. These values are consistent with cleavage C-terminal to residues 238, 232, 217, 190, and the MBP portion of the fusion construct, respectively. Predicted masses for the corresponding MBP-RAG1 polypeptides are 60,897 Da (MBP-RAG1(87-238)), 60,199 Da (MBP-RAG1(87-232)), 58,400 Da (MBP-RAG1(87-217)), and 55,240 Da (MBP-RAG1(87-190)). (E) SEC of MBP-CND following subcloning and purification. (F) MALDI-TOF spectrum of MBP-CND. The peaks yield MWt values consistent with the +1 (1) and +2 (2) species of the protein. (G) SEC of MBP-bZDD following subcloning and purification. All SEC runs were performed using Superdex 200 columns with either 20 ml (panel B) or 200 ml (panels C, E, and G) column volumes.
Arbuckle et al. BMC Biochemistry 2011 12:23 doi:10.1186/1471-2091-12-23