Purification and properties of the ubiquitin-conjugating enzymes Cdc34 and Ubc13.Mms2.
A prerequisite for structure/function studies on the ubiquitin-conjugating enzymes (Ubc) Cdc34 and Ubc13.Mms2 has been the ability to express and purify recombinant derivatives of each. This chapter describes the methods used in the expression and purification of these proteins from Escherichia coli, including variations of these protocols used to generate (35)S, (15)N, (13)C/(15)N, and seleno-L-methionine derivatives. Assays used to measure the Ub thiolester and Ub conjugation activities of these Ubcs are also described.
Ptak
Christopher
C
Department of Biochemistry, University of Utah, Salt Lake City, UT 84103, USA.
Varelas
Xaralabos
X
Moraes
Trevor
T
McKenna
Sean
S
Ellison
Michael J
MJ
D064173:Anaphase-Promoting Complex-Cyclosome; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008025:Ligases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013194:Staining and Labeling; D013462:Sulfur Radioisotopes; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D043743:Ubiquitin-Protein Ligase Complexes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D013462:Sulfur Radioisotopes; C577449:CDC34 protein, S cerevisiae; C577447:CDC34 protein, human; C471610:UBC13 protein, S cerevisiae; C114715:UBE2V2 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D008025:Ligases