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Functional expression of porcine aminoacylase 1 in E. coli using a codon optimized synthetic gene and molecular chaperones

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Efficient recombinant expression of N-acyl-l-aminoacylase 1 from pig kidney (pAcy1) was achieved in the prokaryotic host Escherichia coli. An optimized nucleotide sequence (codon adaptation index 0.95 for E. coli), was cloned into vector pET-52(b) yielding an E. coli-expressible pAcy1 gene. Formation of inclusion bodies was alleviated by co-expression of molecular chaperones resulting in 2.7- and 4.2-fold increased recovery of active pAcy1 using trigger factor or GroEL–GroES, respectively. Facile purification was achieved via StrepTag affinity chromatography. Overall, more than 80 mg highly active pAcy1 (94 U/mg) was obtained per liter of cultivation broth. The protein was analyzed for structural and functional identity, and the performances of further described expression and purification systems for pAcy1 were compared.

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We thank Dr. A. Krager (Friedrich-Loeffler-Institute, Insel Riems, Germany) for mass spectroscopy analysis, F. Leipold for support in CD spectroscopy and the “Fachagentur für Nachwachsende Rohstoffe” (FNR, Gülzow, Germany, Grant No. 22009405) for financial support.

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Correspondence to Uwe Bornscheuer.

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Wardenga, R., Hollmann, F., Thum, O. et al. Functional expression of porcine aminoacylase 1 in E. coli using a codon optimized synthetic gene and molecular chaperones. Appl Microbiol Biotechnol 81, 721–729 (2008). https://doi.org/10.1007/s00253-008-1716-7

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  • N-acyl-l-aminoacylase
  • Synthetic gene
  • Codon usage
  • Soluble expression
  • Inclusion body
  • Chaperones
  • StrepTag