High-level expression, secretion, and purification of the thermostable aqualysin I from Thermus aquaticus YT-1 in Pichia pastoris

Protein Expr Purif. 2003 Jun;29(2):223-9. doi: 10.1016/s1046-5928(03)00060-3.


Aqualysin I is a heat-stable subtilisin-type serine protease which is secreted into the culture medium by Thermus aquaticus YT-1, an extreme thermophile. We report the high-level expression of an aqualysin I protein using its native signal sequence for secretion in the methylotrophic yeast, Pichia pastoris. The expression of aqualysin I in P. pastoris was carried out using the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Pro-aqualysin I (38kDa) as inactive protein was secreted into the medium of shake-flask cultures at a concentration of 1g/L. It was isolated from the culture supernatant by an ammonium sulfate precipitation and one-step anion exchange chromatography in a nearly pure form and was autoproteolytically activiated by heat treatment. A proteolytic activity test indicated that the purified recombinant aqualysin I was properly folded with a specific activity similar to that of the native enzyme. We also explored the possibility of secreting the GAP expressed aqualysin I in P. pastoris by in-frame fusion of the Saccharomyces cerevisiae alpha-factor secretion signal. However, the levels of secreted pro-aqualysin I particles were approximately 10 times lower, possibly as a consequence of membrane association or to the influence of the alpha-factor secretion signal sequences on the transcription or secretion of aqualysin I. When considering further optimization of the downstream process and culture conditions for high-level production of recombinant aqualysin I by P. pastoris, this expression system is promising for development as an industrial process.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • DNA Primers / genetics
  • Enzyme Stability
  • Hot Temperature
  • Molecular Sequence Data
  • Pichia / enzymology*
  • Pichia / genetics
  • Plasmids / genetics
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Serine Endopeptidases / biosynthesis*
  • Serine Endopeptidases / isolation & purification
  • Serine Endopeptidases / metabolism*
  • Thermus / enzymology*
  • Thermus / genetics


  • DNA Primers
  • Recombinant Proteins
  • Serine Endopeptidases
  • aqualysin I