Cloning, characterization and expression of a cDNA clone encoding rabbit ubiquitin-conjugating enzyme, E2(32k)

Abstract

A cDNA clone encoding rabbit E2(32k) was obtained by library screening and PCR. The cDNA contains an open reading frame coding for 238 amino acids which shows an overall identity of 81% to human CDC34, the cell cycle-related ubiquitin-conjugating enzyme. A 50% homology to yeast CDC34 within the conserved core domain was also observed. Northern blot analysis indicated that three transcripts existed in all six rabbit tissues examined but their expression levels varied over a wide range. The putative cDNA coding region was highly expressed in Escherichia coli as a his-tagged protein which was purified to homogeneity. The ability of this expressed protein to form a thiolester bond with ubiquitin showed that it was functionally active, The ability of this protein to catalyze the conjugation of ubiquitin to histone H2A and H2B was also examined.