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Recombinant Tk0522, a carbohydrate esterase homologue from Thermococcus kodakarensis, does not require a signal sequence for translocation to periplasmic space in Escherichia coli

  • Anam Tariq
  • Alina Gul
  • Majida A. Muhammad
  • Naeem RashidEmail author
  • Masood A Siddiqui
Original Article


We have cloned Tk0522, a carbohydrate esterase homologue from Thermococcus kodakarensis, with the deletion of a signal sequence of nineteen amino acids at the N-terminal and produced the recombinant Tk0522 in Escherichia coli. Recombinant Tk0522 was produced in soluble and enzymatically active form. Analysis of the cytosolic, periplasmic and extracellular fractions demonstrated the presence of Tk0522 in cytosolic and periplasmic fractions. The esterase activity in the periplasmic fraction, similar to the cytosolic fraction, increased with the passage of time after induction. No significant amount of esterase activity could be detected in the extracellular fraction. To the best of our knowledge, this is the first report on translocation of a mature archaeal esterase from cytosolic to periplasmic space in E. coli.


Thermococcus kodakarensis Gene expression Recombinant esterase Periplasmic localization 





nano meter


Thermus aqauaticus




gravitational force


Compliance with ethical standards

Conflict of interest

There is no conflict of interest.


  1. Baneyx F (1999) Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol 10:411–421CrossRefGoogle Scholar
  2. Bashir Q, Rashid N, Jamil F, Imanaka T, Akhtar M (2009) Highly thermostable L-threonine dehydrogenase from the hyperthermophilic archaeon Thermococcus kodakaraensis. J Biochem 146:95–102. CrossRefGoogle Scholar
  3. Blight MA, Holland IB (1994) Heterologous protein secretion and the versatile Escherichia coli haemolysin translocator. Trends Biotechnol 12:450–455. CrossRefGoogle Scholar
  4. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (2009) The carbohydrate-active EnZymes database (CAZy): an expert resource for Glycogenomics. Nucleic Acids Res 37:D233–D238. CrossRefGoogle Scholar
  5. Chohan SM, Rashid N (2013) TK1656, a thermostable l-asparaginase from Thermococcus kodakaraensis, exhibiting highest ever reported enzyme activity. J Biosci Bioeng 116:438–443. CrossRefGoogle Scholar
  6. Dalbey RE, Kuhn A (2012) Protein traffic in gram-negative bacteria – how exported and secreted proteins find their way. FEMS Microbiol Rev 36:1023–1045. CrossRefGoogle Scholar
  7. Fukui T, Atomi H, Kanai T, Matsumi R, Fujiwara S, Imanaka T (2005) Complete genome sequence of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 and comparison with Pyrococcus genomes. Genome Res 15:352–363. CrossRefGoogle Scholar
  8. Guo J, Coker AR, Wood SP, Cooper JB, Chohan SM, Rashid N, Akhtar M (2017) Structure and function of the thermostable L-asparaginase from Thermococcus kodakarensis. Acta Crystallogr D Struct Biol 73:889–895. CrossRefGoogle Scholar
  9. Hotta Y, Ezaki S, Atomi H, Imanaka T (2002) Extremely stable and versatile carboxylesterase from a hyperthermophilic archaeon. Appl Environ Microbiol 68:3925–3931. CrossRefGoogle Scholar
  10. Johansson K, El-Ahmad M, Friemann R, Jörnvall H, Markovic O, Eklund H (2002) Crystal structure of plant pectin methylesterase. FEBS Lett 514:243–249CrossRefGoogle Scholar
  11. Jong WS, Sauri A, Luirink J (2010) Extracellular production of recombinant proteins using bacterial autotransporters. Curr Opin Biotechnol 21:646–652CrossRefGoogle Scholar
  12. Lee SY (1996) High cell-density culture of Escherichia coli. Trends Biotechnol 14:98–105CrossRefGoogle Scholar
  13. Makrides SC (1996) Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev 60:512–538Google Scholar
  14. Markovic O, Janecek S (2004) Pectin methylesterases: sequence-structural features and phylogenetic relationships. Carbohydr Res 339:2281–2295CrossRefGoogle Scholar
  15. Morikawa M, Izawa Y, Rashid N, Hoaki T, Imanaka T (1994) Purification and characterization of a thermostable thiol protease from a newly isolated hyperthermophilic Pyrococcus sp. Appl Environ Microbiol 60:4559–4566Google Scholar
  16. Muhammad MA, Falak S, Rashid N, Ahmed N, Gardner QA, Tariq A, Akhtar M (2017) Complete signal peptide of Tk1884, an α-amylase from Thermococcus kodakarensis, is not necessary for extracellular secretion of the enzyme by Escherichia coli. Amylase 1:75–81CrossRefGoogle Scholar
  17. Nisar MA, Rashid N, Bashir Q, Gardner QT, Shafiq MH, Akhtar M (2013) TK1299, a highly thermostable NAD(P)H oxidase from Thermococcus kodakaraensis exhibiting higher enzymatic activity with NADPH. J Biosci Bioeng 116:39–44. CrossRefGoogle Scholar
  18. Ouzzine M, Magdalou J, Burchell B, Fournel-Gigleux S (1999) An internal signal sequence mediates the targeting and retention of the human UDP-glucuronosyltransferase 1A6 to the endoplasmic reticulum. J Biol Chem 274:31401–31409CrossRefGoogle Scholar
  19. Rashid N, Imanaka H, Kanai T, Fukui T, Atomi H, Imanaka T (2002) A novel candidate for the true fructose-1,6-bisphosphatase in archaea. J Biol Chem 277:30649–30655. CrossRefGoogle Scholar
  20. Rosano GL, Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 5:172. Google Scholar
  21. Shokri A, Sandén AM, Larsson G (2003) Cell and process design for targeting of recombinant protein into the culture medium of Escherichia coli. Appl Microbiol Biotechnol 60:654–664. CrossRefGoogle Scholar
  22. Sorensen HP, Mortensen KK (2015) Advanced genetic strategies for recombinant protein expression in Escherichia coli. J Biotechnol 115:113–128CrossRefGoogle Scholar
  23. Tayyab M, Rashid N, Akhtar M (2011) Isolation and identification of lipase producing thermophilic Geobacillus sp. SBS-4S: cloning and characterization of the lipase. J Biosci Bioeng 111:272–278. CrossRefGoogle Scholar
  24. Varejão N, De-Andrade RA, Almeida RV, Anobom CD, Foguel D, Reverter D (2018) Structural mechanism for the temperature-dependent activation of the hyperthermophilic Pf2001 esterase. Structure 26:199–208.e3. CrossRefGoogle Scholar
  25. Zhang JT, Lee CH, Duthie M, Ling V (1995) Topological determinants of internal transmembrane segments in P-glycoprotein sequences. J Biol Chem 270:1742–1746Google Scholar
  26. Zhu Y, Li J, Cai H, Ni H (2013) Characterization of a new thermostable esterase from metagenomic library. Microbiol Res 168:589–597CrossRefGoogle Scholar

Copyright information

© Institute of Molecular Biology, Slovak Academy of Sciences 2019

Authors and Affiliations

  1. 1.School of Biological SciencesUniversity of the PunjabLahorePakistan
  2. 2.Department of ChemistryUniversity of BalochistanQuettaPakistan

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