Optimization of the wheat puroindoline-a production in Pichia pastoris.
OBJECTIVE
A recombinant puroindoline-a (rPIN-a) was produced using the methylotrophic yeast Pichia pastoris.


RESULTS
In fed-batch culture, the production of rPIN-a decreased after 24 h of methanol induction. Most of the rPIN-a was not soluble in the culture medium remaining bound to the cell walls. Soluble and membrane-bound rPIN-a were quantified by ELISA after Triton X-114 phase partitioning. In order to improve the production of rPIN-a, the influence of pH, specific growth rate and the addition of TX-114 was tested on two independent continuous cultures. The production of rPIN-a was improved when continuous culture was carried out at 29 degrees C under acid conditions (pH 5) with a low dilution rate (D=0.025 h(-1)). The addition of 0.01% TX-114 to the medium inverted the ratio between the secreted and the membrane-bound rPIN-a.


CONCLUSIONS
When a continuous culture was carried out under optimized conditions, the rPIN-a production yield was increased 10-fold to 14 mg l(-1) and 80% of the rPIN-a was soluble.


CONCLUSIONS
This study would be helpful to optimize the expression of other membrane-bound proteins in P. pastoris.
Issaly
N
N
UFR Microbiologie Industrielle et de Génétique des Microorganismes, ENSA.M-INRA, Montpellier, France.
Solsona
O
O
Joudrier
P
P
Gautier
M F
MF
Moulin
G
G
Boze
H
H
D003470:Culture Media / Q000737:chemistry; D005285:Fermentation; D005822:Genetic Vectors; D010843:Pichia / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D014908:Triticum / Q000737:chemistry*
D003470:Culture Media; D010940:Plant Proteins; C082942:puroindoline protein, Triticum aestivum