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Related Experiment Videos

Rabbit ubiquitin-activating enzyme E1: cDNA cloning, sequence and expression

B Sun1, K Jeyaseelan, M C Chung

  • 1Department of Biochemistry, Faculty of Medicine, National University of Singapore, Singapore.

Gene
|October 10, 1997
PubMed
Summary
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Researchers isolated and sequenced a rabbit heart cDNA clone for ubiquitin-activating enzyme E1 (E1). The functional recombinant protein showed high homology to other E1 enzymes, confirming its biological activity.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Ubiquitin-activating enzyme E1 (E1) is crucial for the ubiquitin-proteasome system.
  • Understanding E1 enzyme function across species aids in elucidating cellular regulation.

Purpose of the Study:

  • To isolate and characterize the cDNA encoding rabbit ubiquitin-activating enzyme E1.
  • To express and verify the functionality of recombinant rabbit E1 protein.

Main Methods:

  • cDNA library screening and sequencing.
  • Northern blot analysis for gene expression.
  • Recombinant protein expression (His-tagged).
  • Western blot and enzymatic assays for verification.

Main Results:

Related Experiment Videos

  • A 3.485 kb cDNA encoding a 1058 amino acid E1 protein (approx. 118 kDa) was identified.
  • High sequence homology was observed between rabbit E1 and E1 enzymes from other organisms.
  • Northern blot confirmed a 3.5 kb transcript in various rabbit tissues.
  • Recombinant rabbit E1 protein demonstrated cross-reactivity with anti-human E1 antibodies and was enzymatically functional in ubiquitin thiolester assays.

Conclusions:

  • The study successfully isolated and characterized the rabbit E1 cDNA.
  • The recombinant rabbit E1 protein is functional and shares significant homology with orthologs, supporting its role in the ubiquitin conjugation pathway.