Affinity chromatography of native SUMO proteins using His-tagged recombinant UBC9 bound to Co2+-charged talon resin.
Four small ubiquitin-related modifier (SUMO) genes have been identified in humans. The recently identified SUMO4 was detected in mRNA transcripts from HEK293 cells, and human kidney and spleen tissue and may be involved in regulation of NF-kappaB and susceptibility to autoimmune diseases. However, identification and characterization of a native SUMO4 protein has not yet been reported. Here, we analyzed for the presence of native SUMO proteins in HEK293 cells and human kidney tissue using an affinity purification procedure using a UBC9 matrix followed by mass spectroscopy analyses for SUMO-specific peptides. Identification by mass spectroscopy of peptides generated by Trypsin and Lys-C digestion did reveal peptides unique to SUMO1 and SUMO2/3, but not SUMO4. In control experiments, SUMO4 prepared by recombinant methods was isolated and even enriched by our UBC9 affinity purification. Thus, SUMO4 protein appears to be either in extremely low abundance in human kidney or HEK293 cells or it is not present at all. It remains possible that SUMO4 protein is more abundant in other cell types or can be induced by hormonal or environmental challenges and the procedures reported here should be extremely useful for detecting native SUMO4. Furthermore, using His-tagged recombinant proteins bound to Co(2+)-charged Talon resin has general applicability to isolate native proteins that have strong non-covalent interactions with the resin-bound His-tagged proteins.
Bohren
Kurt M
KM
Molecular Diabetes and Metabolism Section and the Harry B. and Aileen B. Gordon Diabetes Research Center, Department of Pediatrics, One Baylor Plaza, Baylor College of Medicine, Houston, TX 77030, USA.
Gabbay
Kenneth H
KH
Owerbach
David
D
D015110:4-Hydroxycoumarins / Q000737:chemistry*; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods*; D002852:Chromatography, Ion Exchange; D003035:Cobalt / Q000737:chemistry*; D006801:Humans; D025841:Small Ubiquitin-Related Modifier Proteins / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry*; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D015110:4-Hydroxycoumarins; C489647:SUMO4 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D003035:Cobalt; C013418:bromfenacoum; D044763:Ubiquitin-Conjugating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9