Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

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

  • 329 Accesses

  • 6 Citations

Abstract

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.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

References

  1. Baneyx F (1999) Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol 10:411–421

  2. Birnbaum SM, Levintow L, Kingsley RB, Greenstein JP (1952) Specificity of amino acid acylases. J Biol Chem 194:455–470

  3. Bommarius AS, Drauz K, Klenk H, Wandrey C (1992) Operational stability of enzymes. Acylase-catalyzed resolution of N-acetyl amino acids to enantiomerically pure l-amino acids. Ann N Y Acad Sci 672:126–136

  4. Bornscheuer UT, Pohl M (2001) Improved biocatalysts by directed evolution and rational protein design. Curr Opin Chem Biol 5:137–143

  5. Chung CT, Niemela SL, Miller RH (1989) One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc Natl Acad Sci U. S. A 86:2175–2172

  6. Dean KES, Klein GR, Renaudet O, Reymond J-L (2003) A green fluorescent chemosensor for amino acids provides a versatile high-throughput screening (HTS) assay for proteases. Bioorg Med Chem Lett 13:1653–1656

  7. Durand A, Giardina T, Villard C, Roussel A, Puigserver A, Perrier J (2003) Rat kidney acylase I: further characterisation and mutation studies on the involvement of Glu 147 in the catalytic process. Biochimie 85:953–962

  8. Fuhrmann M, Hausherr A, Ferbitz L, Schödl T, Heitzer M, Hegemann P (2004) Monitoring dynamic expression of nuclear genes in Chlamydomonas reinhardtii by using a synthetic luciferase reporter gene. Plant Mol Biol 55:869–881

  9. Gentzen I, Löffler HG, Schneider F (1980) Aminoacylase from Aspergillus oryzae. Comparison with the pig kidney enzyme. Z Naturf Sect C: Biosciences 35:544–550

  10. Jana S, Deb JK (2005) Strategies for efficient production of heterologous proteins in Escherichia coli. Appl Microbiol Biotechnol 67:289–298

  11. Kandror O, Sherman M, Rhode M, Goldberg AL (1995) Trigger factor is involved in GroEL-dependent protein degradation in Escherichia coli and promotes binding of GroEL to unfolded proteins. EMBO J 14:6021–6027

  12. Kördel W, Schneider F (1976) Chemical investigations on pig kidney aminoacylase. Biochim Biophys Acta 445:446–457

  13. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

  14. Liu Z, Zhen Z, Zuo Z, Wu Y, Liu A, Yi Q, Li W (2006) Probing the catalytic center of porcine aminoacylase 1 by site-directed mutagenesis, homology modeling and substrate docking. J Biochem 139:421–430

  15. Lugay JC, Aronson JN (1969) Palo Verde (Parkinsonia aculeata L.) seed aminoacylase. Biochim Biophys Acta 191:397–414

  16. Maier T, Drapal N, Thanbichler M, Böck A (1998) Strep-tag II affinity purification: an approach to study intermediates of metalloenzyme biosynthesis. Anal Biochem 259:68–73

  17. Makrides SC (1996) Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev 60:512–538

  18. Mitta M, Ohnogi H, Yamamoto A, Kato I, Sakiyama F, Tsunasawa S (1992) The primary structure of porcine aminoacylase 1 deduced from cDNA sequence. J Biochem 112:737–742

  19. Mitta M, Kato I, Tsunasawa S (1993) The nucleotide sequence of human aminoacylase-1. Biochim Biophys Acta 1174:201–203

  20. Nishihara K, Kanemori M, Kitagawa M, Yanagi H, Yura T (1998) Chaperone coexpression plasmids: differential and synergistic roles of DnaK-DnaJ-GrpE and GroEL-GroES in assisting folding of an allergen of Japanese cedar pollen, Cryj2, in Escherichia coli. Appl Environ Microbiol 64:1694–1699

  21. Novy R (2001) Overcoming the codon bias of E. coli for enhanced protein expression. Newsletter of Novagen:1–3

  22. Palm GJ, Röhm KH (1995) Aminoacylase I from porcine kidney: identification and characterization of two major protein domains. J Protein Chem 14:233–240

  23. Parker KC, Garrels JI, Hines W, Butler EM, McKee AH, Patterson D, Martin S (1998) Identification of yeast proteins from two-dimensional gels: working out spot cross-contamination. Electrophoresis 19:1920–1932

  24. Perkins DN, Pappin DJ, Creasy DM, Cottrell JS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:3551–3567

  25. Pittelkow S, Lindner H, Röhm K-H (1998) Human and porcine aminoacylase I overproduced in a baculovirus expression vector system: evidence for structural and functional identity with enzymes isolated from kidney. Protein Expr Purif 12:269–276

  26. Sakanyan V, Desmarez L, Legrain C, Charlier D, Mett I, Kochikyan A, Savchenko A, Boyen A, Falmagne P, Pierard A (1993) Gene cloning, sequence analysis, purification, and characterization of a thermostable aminoacylase from Bacillus stearothermophilus. Appl Environ Microbiol 59:3878–3888

  27. Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press.

  28. Sato T, Tosa T (1993) Optical resolution of racemic amino acids by aminoacylase. Bioproc Technol 16:3–14

  29. Sharp PM, Li WH (1987) The codon Adaptation Index—a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Res 15:1281–1295

  30. Wada E, Handa M, Imamura K, Sakiyama T, Adachi S, Matsuno R, Nakanishi K (2002) Enzymatic synthesis of N-acyl-l -amino acids in a glycerol–water system using acylase I from pig kidney. J Am Oil Chem Soc 79:41–46

  31. Wang HR, Zhang T, Wang ZF, Wang XC, Zhou HM (1995) Aminoacylase from pig kidney contains no disulfide bonds. Sci China Ser B 38:1454–1448

  32. Wu G, Bashir-Bello N, Freeland SJ (2006) The Synthetic Gene Designer: a flexible web platform to explore sequence manipulation for heterologous expression. Protein Expr Purif 47:441–445

  33. Yang JT, Wu CS, Martinez HM (1986) Calculation of protein conformation from circular dichroism. Methods Enzymol 130:208–269

  34. Yang Y, Wang HR, Zhou HM (1996) Kinetics of inhibition of aminoacylase activity by dithiothreitol or 2-mercaptoethanol. Int J Pept Prot Res 48:532–538

  35. Yoshimune K, Ninomiya Y, Wakayama M, Moriguchi M (2004) Molecular chaperones facilitate the soluble expression of N-acyl-d-amino acid amidohydrolases in Escherichia coli. J Ind Microbiol Biotechnol 31:421–426

  36. Youshko MI, van Rantwijk F, Sheldon RA (2001) Enantioselective acylation of chiral amines catalysed by aminoacylase I. Tetrahedron: Asymmetry 12:3267–3271

Download references

Acknowledgements

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.

Author information

Correspondence to Uwe Bornscheuer.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

Keywords

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