Molecular cloning and characterization of cDNA encoding a ubiquitin-conjugating enzyme from Clonorchis sinensis.
The ubiquitin-proteasome system is an essential mechanism for protein degradation in eukaryotes. Protein ubiquitination is composed of a series of enzymatic reactions. The ubiquitin-conjugating enzyme (E2) is one of the important enzymes involved in the process. A cDNA encoding an E2 enzyme was cloned from a Clonorchis sinensis cDNA library by large-scale sequencing. This new cDNA contains 862 bp with a putative open reading frame of 156 amino acids. The deduced amino acid sequence is 77% identical to the human E2, HHR6A and HHR6B. The coding region of this cDNA was expressed in E. coli as a GST-tagged protein, and was purified to electrophoretic homogeneity. Enzymatic assays showed that this E2 had the capacity to form a thiolester linkage, and could conjugate ubiquitin to histone H2A in an E3-independent manner in vitro, which indicated that the expressed protein was functionally active. The nucleotide sequence reported in this paper has been submitted to the Genbank Database with accession number AY632078.
Song
Linxia
L
Department of Parasitology, Medical School, Sun Yat-Sen University, 510089, Guangzhou, PR China.
Chen
Shouyi
S
Yu
Xinbing
X
Wu
Zhongdao
Z
Xu
Jin
J
Yang
Guang
G
Zheng
Nancai
N
Hu
Xuchu
X
Guo
Lingchen
L
Dai
Jianfeng
J
Xu
Jian
J
Ji
Chaoneng
C
Gu
Shaohua
S
Ying
Kang
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003004:Clonorchis sinensis / Q000201:enzymology* / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D018509:DNA, Helminth / Q000235:genetics*; D015723:Gene Library; D017238:Genes, Helminth; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D018509:DNA, Helminth; D011994:Recombinant Proteins; D044763:Ubiquitin-Conjugating Enzymes