Functional expression of amyloidogenic human stefins A and B in Pichia pastoris using codon optimization

Biotechnol Appl Biochem. 2013 May-Jun;60(3):283-8. doi: 10.1002/bab.1079. Epub 2013 May 8.

Abstract

Complementary DNAs encoding human stefins A (HSA) and B (HSB) were synthesized using Pichia-preferred codons by overlap extension PCR. The full-length genes were ligated downstream of the glyceraldehyde-3-phosphate dehydrogenase promoter in the Pichia expression vector pGAPZαC and successfully expressed in Pichia pastoris strain X-33. Functional recombinant HSA and HSB proteins were purified from culture medium at yields of 121.3 ± 13.5 (n = 3) and 95.4 ± 4.1 (n = 3) mg/L, respectively. Using this expression strategy, we demonstrated that high levels of bioactive recombinant HSA and HSB can be produced by fermentation in P. pastoris.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amyloidogenic Proteins / genetics*
  • Amyloidogenic Proteins / metabolism
  • Base Sequence
  • Codon / genetics*
  • Cystatin A / genetics*
  • Cystatin A / metabolism
  • Cystatin B / genetics*
  • Cystatin B / metabolism
  • DNA, Complementary / genetics*
  • Fermentation / genetics
  • Genetic Vectors / genetics
  • Glyceraldehyde-3-Phosphate Dehydrogenases / genetics
  • Glyceraldehyde-3-Phosphate Dehydrogenases / metabolism
  • Humans
  • Molecular Sequence Data
  • Pichia / genetics*
  • Pichia / metabolism
  • Promoter Regions, Genetic / genetics
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism

Substances

  • Amyloidogenic Proteins
  • CSTB protein, human
  • Codon
  • Cystatin A
  • DNA, Complementary
  • Recombinant Proteins
  • CSTA protein, human
  • Cystatin B
  • Glyceraldehyde-3-Phosphate Dehydrogenases