Biomedical Engineering Reference
In-Depth Information
well as identifying relevant structural boundaries to provide for proper protein folding
and stability [44].
3.3.3 Modification of P. pastoris Host Strains for Overexpression
of Protein Disulfide Isomerase
Another area that has been evaluated to increase recombinant protein production in
P. pastoris is the augmentation or overexpression of a multifunctional chaperone in
the endoplasmic reticulum identified as protein disulfide isomerase. In the P. pastoris
expression system, recombinant proteins are commonly targeted for secretion to the
extracellular media. These proteins are translocated into the endoplasmic reticulum,
where folding takes place before secretion through the Golgi apparatus. These proteins
must pass a quality control checkpoint before being exported [45]. In the case of a strong
expression system like P. pastoris, the disulfide exchange machinery may be either
overloaded or inefficient in folding high levels of complex proteins. Both cases result in
low yield of recombinant protein. Engineering the P. pastoris production clone for an
increase in both quantity and quality of drug substance adheres to a QbD approach.
PDI is a multifunctional protein that participates in protein folding, assembly, and
posttranslational modification in the endoplasmic reticulum. It is capable of functioning
both as an enzyme and as a chaperone. PDI catalyzes both the oxidation and isomeriza-
tion of disulfides of nascent polypeptides. As an enzyme, it increases the rate of disulfide
bond formation without altering the folding pathway [46]. While serving as a chaperone,
it promotes correct folding of proteins by preventing the misfolding and aggregation of
partially folded or misfolded peptides [46]. Elevated levels of PDI activity in bacterial,
yeast, and insect cell expression systems increase secretion of heterologous proteins with
or without disulfide bonds [47-50].
We evaluated whether overproduction of P. pastoris PDI (PpPDI) or a PDI
orthologue identified in P. falciparum (PfPDI) when overexpressed would increase
the production level of a transmission blocking vaccine candidate Pfs25, which
contained 11 disulfide bonds and was expressed in P. p a s t o r i s using a synthetic
P. pastoris codon optimized gene [51]. The results from this study have been reported
andarebrieflysummarizedhere[12].BothPpPDIandPfPDIwereclonedintothe
pPICZ
A expressionvector using either the native gene or yeast codon optimized gene
that was transformed into a P. p a s t o r i s production clone that expressed the heterolo-
gous P. falciparum Pfs25 protein. The results of a head-to-head comparison using 5L
bioreactors with defined media are shown in Fig. 3.9. Analysis of fermentation
supernatant showed the benefit of overexpression of PpPDI and a moderate benefit
of the PfPDI (Fig. 3.9). Analysis of the product concentration within the supernatant
by high-performance liquid chromatography method demonstrated that overproduc-
tion of PpPDI increased the level of Pfs25 protein production by three- to fourfold.
Interestingly, not only the quantity of the product was affected but also the quality of
the product was significantly improved, as demonstrated by the subsequent
analysis of the final bulk protein by electron spray ionization mass spectroscopy. As
discussed earlier, malaria proteins lack significant glycosylation [35]. Therefore,
the reduction in the level of O-linked mannosylation on Pfs25 due to PpPDI
a
