Secretion and assembly of calicivirus-like particles in high-cell-density yeast fermentations: strategies based on a recombinant non-specific BPTI-Kunitz-type protease inhibitor.
The yeast Pichia pastoris is one of the most robust cell factories in use for the large-scale production of biopharmaceuticals with applications in the fields of human and animal health. Recently, intracellular high-level expression of rabbit hemorrhagic disease virus (RHDV) capsid protein (VP1) as a self-assembled multipurpose antigen/carrier was established as a production process from P. pastoris. Since recovery of VP1 from the culture media implies technological and economic advantages, the secretion of VP1 variants was undertaken in this work. Conversely, extensive degradation of VP1 was detected. Variations to culture parameters and supplementation with different classes of additives were unable to diminish degradation. Strategies were then conducted during fermentations using a recombinant variant of a non-specific BPTI-Kunitz-type protease inhibitor (rShPI-1A) isolated from the sea anemone Stichodactyla helianthus. The presence of the inhibitor in the culture medium at the recombinant protein induction phase, as well as co-culture of the yeast strains expressing VP1 and rShPI-1A, led to VP1 protection from proteolysis and to production of ordered virus-like particles. A yeast strain was also engineered to co-express the rShPI-1A inhibitor and intact VP1. Expression levels up to 116 mg L(-1) of VP1 were reached under these approaches. The antigen was characterized and purified in a single chromatography step, its immunogenic capacity was evaluated, and a detection test for specific antibodies was developed. This work provides feasible strategies for improvements in P. pastoris heterologous protein secretion and is the first report on co-expression of the ShPI-1A with a recombinant product otherwise subjected to proteolytic degradation.
Animal Biotechnology Division, Center for Genetic Engineering and Biotechnology (CIGB), P.O. Box 6162, 10600, Havana, Cuba.
Erlinda
E
Fernández
Jorge R
JR
Toledo
Manuel
M
Mansur
Oliberto
O
Sánchez
Dayrom F
DF
Gil
Yamile
Y
González-González
Emilio
E
Lamazares
Yaiza
Y
Fernández
Francisco
F
Parra
Omar
O
Farnós
D000818:Animals; D005285:Fermentation; D017929:Hemorrhagic Disease Virus, Rabbit / Q000235:genetics*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011480:Protease Inhibitors / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D012615:Sea Anemones / Q000235:genetics; D015678:Viral Structural Proteins / Q000235:genetics / Q000378:metabolism*; D022701:Virosomes / Q000235:genetics / Q000378:metabolism*
D011480:Protease Inhibitors; D011994:Recombinant Proteins; D015678:Viral Structural Proteins; D022701:Virosomes