Isolation of a cDNA encoding a mammalian multiubiquitinating enzyme (E225K) and overexpression of the functional enzyme in Escherichia coli

Zhijian Chen, Edward G. Niles, Cecile M. Pickart

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Abstract

The ubiquitin (Ub)-conjugating enzyme E225K catalyzes the synthesis of multi-Ub chains in which successive Ub units are linked by an isopeptide bond involving the ε-amino group of Lys-48 of Ubn, and the COOH-terminal Gly residue of Ubn+1 (Chen, Z., and Pickart, C. M. (1990) J. Biol. Chem., 265, 21835-21842). We now describe the polymerase chain reaction (PCR)-based cloning of an E225K-encoding cDNA from a bovine thymus library, using degenerate oligonucleotide primers based on the sequences of two E225K peptides. The cDNA encodes a 200-residue protein whose sequence bears similarities of 66 and 59%, respectively, to the sequences of the Ub-conjugating enzymes encoded by the UBC1 and UBC4/UBC5 genes of the yeast Saccharomyces cerevisiae. These three yeast E2s play key roles in Ub-dependent proteolysis (Seufert, W., McGrath, J. P., and Jentsch, S. (1990) EMBO J. 9, 4535-4541). Comparison of the amino acid sequence of E225K with other known E2 sequences strongly suggests that Cys-92, one of two E225K Cys residues, forms the Ub thiol ester adduct that is an intermediate in E2-catalyzed multiubiquitination. The E225K-encoding cDNA was overexpressed in Escherichia coli, and the recombinant E225K protein was purified to electrophoretic homogeneity; enzymatic assays showed that its multiubiquitinating activity was quantitatively identical with that of the native protein. The availability of a cloned cDNA will allow us to assess the physiological role of E225K.

Original languageEnglish (US)
Pages (from-to)15698-15704
Number of pages7
JournalJournal of Biological Chemistry
Volume266
Issue number24
StatePublished - 1991

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Ubiquitin
Escherichia coli
Complementary DNA
Ubiquitin-Conjugating Enzymes
Yeast
Enzymes
Yeasts
Proteolysis
Thymus
DNA Primers
Cloning
Polymerase chain reaction
Enzyme Assays
Recombinant Proteins
Sulfhydryl Compounds
Thymus Gland
Saccharomyces cerevisiae
Organism Cloning
Amino Acid Sequence
Assays

ASJC Scopus subject areas

  • Biochemistry

Cite this

Isolation of a cDNA encoding a mammalian multiubiquitinating enzyme (E225K) and overexpression of the functional enzyme in Escherichia coli. / Chen, Zhijian; Niles, Edward G.; Pickart, Cecile M.

In: Journal of Biological Chemistry, Vol. 266, No. 24, 1991, p. 15698-15704.

Research output: Contribution to journalArticle

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