OPTIMISATION OF RECOMBINANT PROTEIN PRODUCTION IN PICHIA PASTORIS, ACTA UNIVERSITATIS OULUENSIS C Technica 393
|Kustantaja:||Oulun yliopisto|| |
|Oppiaine:||Tekniikka, matematiikka|| |
|Sijainti:||Print Tietotalo|| |
|Tekijät:||KHATRI NARENDAR KUMAR || |
Potential lethal diarrhoea caused by enterotoxigenic Escherichia coli strains is one of the mostcommon diseases in young pigs. It can be cured by single-chain antibody fragments (scFv), whichcan be produced in recombinant microorganisms. Pichia pastoris, a methylotrophic yeast, isgenerally considered an interesting production system candidate, as it can secrete properly foldedproteins. These proteins accumulate in high concentrations during fermentation, reducing the costfor product recovery.
Strong inducible AOX1 promoter, widely used in P. pastoris for fast, inexpensive production,is typically induced by methanol. The high oxygen demand of methanol metabolism makesoxygen supply a major parameter in cultivations requiring special process design strategies. Instandard fed-batch cultivation, dissolved oxygen concentration inside a bioreactor is kept at acertain level by pumping air and pure oxygen into the reactor. There are safety concerns over thehandling of oxygen, especially at a large scale. Therefore, there is a need to develop a productionprocess under oxygen-limited conditions.
This dissertation studies the development of a cost-efficient production process of scFv in P.pastoris. Both methanol and oxygen parameters influence the production process and the objectivewas to find a robust production process. Fed-batch cultivations were performed in a 10 L scalebioreactor. The effects of lower oxygen level, methanol concentration, glycerol feeding durationand specific substrate-uptake rates on product formation were studied. A P. pastoris GS115 his4strain under an AOX1 promoter system expressing scFv was used in this study. The fed-batchfermentations were carried out in a bioreactor with basal salt media.
In this doctoral dissertation, a process was developed for a single-chain antibody fragment(scFv) production in P. pastoris. The product levels of 3.5 g L-1 scFv in culture supernatant wereachieved and a production process was designed without additional need of pure oxygen, thusrelieving safety requirements and lowering the amount of methanol. The process developed duringthis research may potentially be utilised by both academia and industry having interests inexpressing proteins in P. pastoris. The methanol-uptake control strategy is beneficial for thoseproducts that suffer from degradation or modification during limited feeding of methanol.