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Related Experiment Videos

Expression of Carcinoscorpius rotundicauda factor C cDNA

S D Roopashree1, C Chai, B Ho

  • 1Department of Zoology, National University of Singapore, Kent Ridge Crescent.

Biochemistry and Molecular Biology International
|April 1, 1995
PubMed
Summary

The horseshoe crab Factor C (FC) gene

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Factor C (FC) is a key component of the horseshoe crab clotting cascade.
  • Understanding the expression of FC is crucial for its applications in diagnostics.

Purpose of the Study:

  • To investigate the effect of 5' untranslated regions (UTR) on the in vitro and in vivo expression of Carcinoscorpius rotundicauda Factor C (CrFC).
  • To compare the expression efficiency of different CrFC cDNA constructs.

Main Methods:

  • In vitro coupled transcription-translation (TnT) using T7 promoter.
  • Analysis of full-length cDNA (CrFC26, CrFC21) and deletion mutants.
  • Comparison of in vitro and in vivo (E. coli) expression of truncated CrFC21-T7 gene fusions.

Main Results:

  • Full-length CrFC21 with an accessible ATG codon yielded high levels of FC in vitro.
  • Wild-type CrFC26 with a long 5' UTR and multiple false ATGs failed to produce a translated product.
  • CrFC26 deletion mutants and truncated CrFC21 constructs showed translatability but at lower efficiency than CrFC21.
  • In vitro expression yielded single products, while E. coli produced three major immunoreactive FC bands.

Conclusions:

  • The 5' UTR significantly impacts the translational efficiency of horseshoe crab Factor C.
  • CrFC21 is a more suitable construct for high-yield recombinant FC production.
  • Differences in expression patterns between in vitro and in vivo systems highlight the complexity of gene expression regulation.

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