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Codon optimisation is key for pernisine expression in Escherichia coli.

Marko Šnajder1, Marko Mihelič2, Dušan Turk2

  • 1Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.

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|April 10, 2015
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Summary

Codon optimization enabled overexpression of the hyperthermophilic protease pernisine in E. coli. The recombinant enzyme, pernisineco, is active at 100°C and pH 7.0, retaining native characteristics.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Pernisine is an extracellular serine protease from the hyperthermophilic Archaeon Aeropyrum pernix K1.
  • Low yields and expression issues limit industrial applications of native pernisine.

Purpose of the Study:

  • To overcome challenges in pernisine overexpression and characterize its recombinant form.
  • To investigate the role of a putative active site serine residue (S355) in pernisine activity and activation.

Main Methods:

  • Codon optimization and de novo DNA synthesis for pernisine gene expression in E. coli.
  • Cloning of wild-type, codon-optimized, and mutant pernisine genes into expression vectors.
  • Purification of recombinant pernisine using chromatography techniques.
  • Confirmation of protein identity via N-terminal sequencing, mass spectrometry, and immunodetection.
  • Enzymatic activity assays under varying temperature and pH conditions.

Main Results:

  • Codon-optimized pernisine (pernisineco) and a mutant form (pernisineS355Aco) were successfully expressed and purified as 55-kDa proforms from E. coli at approximately 10 mg/L yield.
  • Wild-type pernisine (pernisinewt) showed no detectable expression.
  • Mature pernisineco exhibited proteolytic activity (36 kDa) after heat activation, while pernisineS355Aco remained inactive.
  • Optimal activity for recombinant pernisine was observed at approximately 100°C and pH 7.0.

Conclusions:

  • Codon optimization is essential for efficient pernisine overexpression in E. coli.
  • The catalytic Ser355 residue is critical for pernisine activity but not for its autoproteolytic activation.
  • Recombinant pernisine retains the characteristics of the native enzyme, functioning as a calcium-modulated, thermostable serine protease.