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

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 pGAPZC 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.