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Cloning and heterologous expression of bovine pyroglutamyl peptidase type-1 in Escherichia coli: purification, biochemical and kinetic characterisation

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Abstract

We describe the cloning, expression and purification of the bovine XM866409 form of pyroglutamyl peptidase type-1 (PAP1). The cloned nucleotide sequence has an ORF coding for a primary sequence of 209 amino acid residues, which displays 98% identity with the human AJ278828 form of the enzyme. Three amino acid residues at positions 81, 205 and 208 were found to vary between the two sequences. The recombinant bovine PAP1 with a C-terminal His6 tag (rBtaPAP16H) was expressed in Escherichia coli XL10-Gold cells and purified by immobilised nickel ion affinity chromatography resulting in a yield of 2.6 mg of PAP1 per litre of culture. Purified rBtaPAP16H had a specific activity of 3633 units mg−1. SDS-PAGE revealed a band for bovine PAP1 with a molecular weight of ∼24 kDa, which is in good agreement with previously reported data on PAP1. The K m and k cat values obtained for rBtaPAP16H were 59 μM and 3.5 s−1, respectively. The optimum pH for activity was 9.0–9.5 and the optimum temperature was 37 °C. rBtaPAP16H was found to have an absolute requirement for the thiol-reducing agent DTT, consistent with the expected property of a cysteine protease. Kinetic studies using the peptides pGlu-His-Pro-NH2 (TRH), pGlu-Ala and pGlu-Val revealed K i values of 44.1, 141 and 652.17 μM, respectively. The lowest K i, observed for Thyrotropin-releasing Hormone (TRH), indicates that rBtaPAP16H has a higher affinity for tripeptides over dipeptides.

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Abbreviations

AMC:

7-amino-4-methylcoumarin

BCA:

Bicinchoninic acid

DMSO:

Dimethyl sulphoxide

DTT:

Dithiothreitol

IPTG:

Isopropyl-β-d-thiogalactopyranoside

LH-RH:

Luteinising hormone-releasing hormone

PAP:

Pyroglutamyl peptidase

pGlu:

Pyroglutamic acid

TRH:

Thyrotropin-releasing hormone

References

  1. Barrett AJ, Rawlings ND (2001) Evolutionary lines of cysteine peptidases. Biol Chem 382:727–733

    Article  PubMed  CAS  Google Scholar 

  2. Doolittle RF, Armentrout RW (1968) Pyrrolidonyl peptidase. An enzyme for selective removal of pyrrolidonecarboxylic acid residues from polypeptides. Biochemistry 7:516–521

    PubMed  CAS  Google Scholar 

  3. Szewczuk A, Mulczyk M (1969) Pyrrolidonyl peptidase in bacteria. The enzyme from Bacillus subtilis. Eur J Biochem 6:63–67

    Article  Google Scholar 

  4. Tsuru D, Fujiwara K, Kado K (1978) Purification and characterization of l-pyrrolidonecarboxylate peptidase from Bacillus amyloliquefaciens. J Biochem (Tokyo) 84:467–476

    CAS  Google Scholar 

  5. Taylor WL, Dixon JE (1978) Characterization of a pyroglutamate aminopeptidase from rat serum that degrades thyrotropin-releasing hormone. J Biol Chem 253:6934–6940

    PubMed  CAS  Google Scholar 

  6. Tsuru D, Sakabe K, Yoshimoto T, Fujiwara K (1982) Pyroglutamyl peptidase from chicken liver: Purification and some properties. J Pharmacobiodyn 5:859–868

    PubMed  CAS  Google Scholar 

  7. Cummins PM, O’Connor B (1998) Pyroglutamyl peptidase: An overview of the three known enzymatic forms. Biochim Biophys Acta 1429:1–17

    PubMed  CAS  Google Scholar 

  8. Irazusta J, Gil J, Ruiz F, Agirregoitia N, Casis L, Silveira PF (2002) Effect of the disruption of body fluid balance on pyroglutamyl aminopeptidase (Type−1) in rat brain structures. Neuropeptides 36:333–340

    Article  PubMed  CAS  Google Scholar 

  9. Falkous G, Harris JB, Mantle D (1995) Effect of neurotoxic metal ions in vitro on proteolytic enzyme activities in human cerebral cortex. Clinice Chimica Acta 238:125

    Article  CAS  Google Scholar 

  10. Faivre-Bauman A, Loudes C, Barret A, Tixier-Vidal A, Bauer K (1986) Possible role of neuropeptide degrading enzymes on thyroliberin secretion in fetal hypothalamic cultures in serum free medium. Neuropeptides 7:125–138

    Article  PubMed  CAS  Google Scholar 

  11. Brunetti L, Cacciatore I, Di Stefano A, Dupre S, Giorgi A, Luisi G, Michelotto B, Orlando G, Pinnen F, Recinella L (2002) Synthesis and biological evaluation of a novel pyroglutamyl-modified TRH analogue. Farmaco 57:479–486

    Article  PubMed  CAS  Google Scholar 

  12. Yoshimoto T, Shimoda T, Kitazono A, Kabashima T, Ito K, Tsuru D (1993) Pyroglutamyl peptidase gene from Bacillus amyloliquefaciens: cloning, sequencing, expression, and crystallization of the expressed enzyme. J Biochem (Tokyo) 113:67–73

    CAS  Google Scholar 

  13. Odagaki Y, Hayashi A, Okada K, Hirotsu K, Kabashima T, Ito K, Yoshimoto T, Tsuru D, Sato M, Clardy J (1999) The crystal structure of pyroglutamyl peptidase I from Bacillus amyloliquefaciens reveals a new structure for a cysteine protease. Structure 7:399–411

    Article  PubMed  CAS  Google Scholar 

  14. Singleton MR, Isupov MN, Littlechild JA (1999) Crystallisation and preliminary X-ray diffraction studies of pyrrolidone carboxyl peptidase from the hyperthermophilic archaeon Thermococcus litoralis. Acta Crystallogr D 55:702–703

    Article  PubMed  CAS  Google Scholar 

  15. Sokabe M, Kawamura T, Sakai N, Yao M, Watanabe N, Tanaka I (2002) The X-ray crystal structure of pyrrolidone-carboxylate peptidase from hyperthermophilic archaea Pyrococcus horikoshii. J Struct Funct Genomics 2:145–154

    Article  PubMed  CAS  Google Scholar 

  16. Tanaka H, Chinami M, Mizushima T, Ogasahara K, Ota M, Tsukihara T,Yutani K (2001) X-ray crystalline structures of pyrrolidone carboxyl peptidase from a hyperthermophile, Pyrococcus furiosus, and its cys-free mutant. J Biochem (Tokyo) 130:107–118

    CAS  Google Scholar 

  17. Dando PM, Fortunato M, Strand GB, Smith TS, Barrett AJ (2003) Pyroglutamyl-peptidase I: Cloning, sequencing, and characterisation of the recombinant human enzyme. Protein Expr Purif 28:111–119

    Article  PubMed  CAS  Google Scholar 

  18. Clarke P: PhD thesis, Dublin City University, Ireland, 2000

  19. Fujiwara K, Tsuru D (1978) New chromogenic and fluorogenic substrates for pyrrolidonyl peptidase. J Biochem (Tokyo) 83:1145–1149

    CAS  Google Scholar 

  20. Browne P, O’Cuinn G (1983) An evaluation of the role of a pyroglutamyl peptidase, a post-proline cleaving enzyme and a post-proline dipeptidyl amino peptidase, each purified from the soluble fraction of guinea-pig brain, in the degradation of thyroliberin in vitro. Eur J Biochem 137:75–87

    Article  PubMed  CAS  Google Scholar 

  21. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 1500:76–85

    Article  Google Scholar 

  22. Lineweaver H, Burk D (1934) Determination of enzyme dissociation constants. J Am Chem Soc 56:658–666

    Article  CAS  Google Scholar 

  23. Eadie GS (1942) The inhibition of cholinesterase by physostigmine and prostigmine. J Biol Chem 146:85–93

    CAS  Google Scholar 

  24. Hanes CS (1932) Studies on plant amylases: The effect of starch concentration upon the velocity of hydrolysis by the amylase of germinated barley. Biochem J 26:1406–1421

    PubMed  CAS  Google Scholar 

  25. Nicholas KB, Nicholas Jr HB, Deerfield II DW (1997) GeneDoc: Analysis and visualization of genetic variation. EMBNEW News 4:14

    Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  27. Gonzalès T, Awadé A (1992) Purification and characterisation of recombinant pyrrolidone carboxyl peptidase of Bacillus subtilis. J Chromatogr 584:101–107

    PubMed  Google Scholar 

  28. Awadé A, Gonzalès T, Cleuziat P, Robert-Baudouy J (1992) One step purification and characterisation of the pyrrolidone carboxyl peptidase of Streptococcus pyogenes over-expressed in Escherischia coli. Fed Eur Biochem Soc 308:70–74

    Google Scholar 

  29. Tsunasawa S, Nakura S, Tanigawa T, Kato I (1998) Pyrrolidone carboxyl peptidase from the hyperthermophilic archaeon Pyrococcus furiosus: Cloning and overexpression in Escherichia coli of the gene, and its application to protein sequence analysis. J Biochem 124:778–783

    PubMed  CAS  Google Scholar 

  30. Singleton M, Taylor S, Parrat J, Littlechild J (2000) Cloning, expression, and characterization of pyrrolidone carboxyl peptidase from the archaeon Thermococcus litoralis. Extremophiles 4:297–303

    Article  PubMed  CAS  Google Scholar 

  31. Cummins PM, O’Connor B (1996) Bovine brain pyroglutamyl aminopeptidase (type-1): Purification and characterisation of a neuropeptide-inactivating peptidase. Int J Biochem Cell Biol 28: 883–893

    Article  PubMed  CAS  Google Scholar 

  32. Awade A, Cleuziat P, Gonzales T, Robert-Baudouy J (1992) Characterization of the pcp gene encoding the pyrrolidone carboxyl peptidase of Bacillus subtilis. FEBS Lett 305:67–73

    Article  PubMed  CAS  Google Scholar 

  33. Tsuru D, Sakabe K, Yoshimoto T. Fujiwara K (1982) Pyroglutamyl peptidase from chicken liver: Purification and some properties. J Pharmacobio Dyn 5:859–868

    PubMed  CAS  Google Scholar 

  34. Lauffart B, Mantle D (1988) Rationalization of aminopeptidase activities in human skeletal muscle soluble extract. Biochim Biophys Acta 956:300–306

    PubMed  CAS  Google Scholar 

  35. Lauffart B, McDermott JR, Biggins JA, Gibson AM, Mantle D (1989) Purification and characterization of pyroglutamyl aminopeptidase from human cerebral cortex. Biochem Soc Trans 17:207–208

    CAS  Google Scholar 

  36. Mantle D, Lauffart B, Gibson A (1991) Purification and characterisation of leucyl aminopeptidase and pyroglutamyl aminopeptidase from human skeletal muscle. Clin Chim Acta 197:35–46

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We thank Prof. Oliver Dolly and the International Centre for Neurotherapeutics and Mayo Vocational Education Committee for funding. We would also like to acknowledge our gratitude to Dr. Paul Clarke for providing us with the pPC225 expression vector, Dr. Ciaran Fagan for use of Enzfitter Software and Dr. Michael Parkinson for help and use of SPPS software.

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Authors and Affiliations

  1. School of Biotechnology, National Centre for Sensor Research, Dublin City University, Dublin 9, Republic of Ireland

    Zelda Kilbane, Páraic Ó Cuív & Brendan O’Connor

  2. International Centre for Neurotherapeutics, Research & Engineering Building, Dublin City University, Dublin 9, Republic of Ireland

    Paul-Roman Vaas

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  1. Zelda Kilbane
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  2. Paul-Roman Vaas
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  3. Páraic Ó Cuív
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  4. Brendan O’Connor
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Correspondence to Zelda Kilbane.

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Kilbane, Z., Vaas, PR., Cuív, P.Ó. et al. Cloning and heterologous expression of bovine pyroglutamyl peptidase type-1 in Escherichia coli: purification, biochemical and kinetic characterisation. Mol Cell Biochem 297, 189–197 (2007). https://doi.org/10.1007/s11010-006-9346-9

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  • DOI: https://doi.org/10.1007/s11010-006-9346-9

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