Optimization of temperature–glycerol–pH conditions for a fed-batch fermentation process for recombinant hookworm (Ancylostoma caninum) anticoagulant peptide (AcAP-5) production by Pichia pastoris

Fermentation strategies for recombinant protein expression in the methylotrophic yeastPichia pastoris

“…Previous studies [7,23,28] revealed that during the batch, fed-batch, or induction phase, the use of glycerol results in ethanol production. Ethanol repression of the AOX1 promoter was investigated using the GS115 (Mut + ) host, expressing intracellular β-galactosidase.…”

“…We have recently applied Response Surface methodology (RSM) to optimize pH, temperature and glycerol feed rate in GFB for the secreted production of hookworm (Ancylostoma caninum) anticoagulant peptide (rAcAP-5) [28]. The RSM is a group of statistical techniques used to evaluate relationships between one or more measured responses and a number of quantitative independent variables that may have an important effect on the measured responses.…”

Expression of Pisum sativum defensin 1 (Psd1) in shaking flasks and bioreactor cultivations of recombinant Pichia pastoris at different pHs

“…However, this variable may affect protease activity and, as a consequence, protein secretion (Sreekrishna et al, 1997;Inan et al, 1999;Files et al, 2001). Based on this and on empirical modeling reported elsewhere (Larentis et al, 2001), three different pH conditions were tested in experiments carried out in shaking flasks.…”

“…The use of Pichia pastoris as an expression system in bioreactors has been described for production of several proteins, such as hepatitis B surface antigen (Cregg et al, 1987), lysozyme (Brierley et al, 1990), glycolate oxidase and catalase (Payne et al, 1995 and, thrombomodulin fragment (Chen et al, 1997), -galactosidase (Chiruvolu et al, 1997), Ancylostoma caninum anticoagulant peptide (Inan et al, 1999), human -galactosidase A (Chen et al, 2000), lipase (Minning et al, 2001), porcine folliclestimulating hormone (Boze et al, 2001), protease inhibitor cystatin C (Files et al, 2001), yielding from hundreds of milligrams to a gram per liter, in timeconsuming processes of up to 200 h. Many of these proteins are currently used as vaccines and human pharmaceuticals, as Pichia pastoris does not produce endotoxins and the growth on medium containing methanol helps to avoid contamination by microorganisms (Cregg et al, 1987;Chen et al, 1997;Cereghino and Cregg, 2000). According to those reports, obtaining a high yield of foreign protein expression seems to depend on rigorous control of methanol level and a high level of maintenance of dissolved oxygen on growth medium.…”

Purification of a recombinant heavy chain fragment C vaccine candidate against botulinum serotype C neurotoxin [rBoNTC(Hc)] expressed in Pichia pastoris

“…Proteolyic degradation of recombinant proteins expressed in P. pastoris can be problematic [19]. Alternative methods such as limiting growth rate [20,21], manipulation of growth temperature [22,23] and addition of protease inhibitors to the fermentation medium have been used to reduce protease activity, but in the case of rBoNTC(H c ) these manipulations proved to be unsuccessful, requiring a different approach. Analysis of in-process purification samples by Western blot and N-terminal sequencing indicated several truncated forms of rBoNTC(H c ).…”