Recombinant Tk0522, a carbohydrate esterase homologue from Thermococcus kodakarensis, does not require a signal sequence for translocation to periplasmic space in Escherichia coli
- 20 Downloads
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.
KeywordsThermococcus kodakarensis Gene expression Recombinant esterase Periplasmic localization
Compliance with ethical standards
Conflict of interest
There is no conflict of interest.
- Makrides SC (1996) Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev 60:512–538Google Scholar
- 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
- 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