Changing the N-terminal sequence protects recombinant Plasmodium falciparum circumsporozoite protein from degradation in Pichia pastoris

Appl Microbiol Biotechnol. 2008 Feb;78(1):139-45. doi: 10.1007/s00253-007-1294-0. Epub 2007 Dec 20.

Abstract

Proteolytic degradation is the primary obstacle in the use of the yeast Pichia pastoris for the expression of recombinant proteins. During the production of a recombinant Plasmodium falciparum circumsporozoite protein in this system, the (NANP)( n ) repeats region at the N-terminus were completely proteolytically degraded. To remove the potential proteolytic site within the recombinant protein, different strategies were tried, including adjusting the cultivation conditions and mutating the sequence at the junction of the repeat domain and C-terminal region, but the degradation continued. However, modification of the N-terminal sequence by adding an epitope-based peptide to the N-terminus not only protected the repeat domain from cleavage by native proteases during longer induction in the yeast host and purification process, but also stabilized this recombinant protein emulsified with adjuvant ISA720 for at least 6 months. The results showed that proteolytic degradation of the recombinant circumsporozoite protein produced in P. pastoris was amino acid sequence (NANP)-specific, and that this effect was likely dependent on the conformation of the recombinant protein.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Emulsions
  • Mannitol / analogs & derivatives
  • Mannitol / chemistry
  • Mannitol / metabolism
  • Mutagenesis, Insertional
  • Oleic Acids / chemistry
  • Oleic Acids / metabolism
  • Pichia / genetics*
  • Pichia / metabolism*
  • Protozoan Proteins / genetics*
  • Protozoan Proteins / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism

Substances

  • Emulsions
  • Oleic Acids
  • Protozoan Proteins
  • Recombinant Proteins
  • circumsporozoite protein, Protozoan
  • mannide monooleate
  • Mannitol