Cultivation of yeast Pichia pastoris in the microtiter plate, for
optimisation of culture conditions, and expression screening of
transformants has gained significance in recent years. However, in the
microtiter plate, it has been challenging to attain cell densities
similar to well-aerated shake-flask culture, due to the poor mixing
resulting in oxygen limitation. To solve this problem, we investigated
the influence of multiple cultivation parameters on P. pastoris
cell growth, including the architecture of 96-deepwell plate (96-DWP),
shaking throw diameter, shaking frequency, culture volume/well, and
media composition. In the optimised conditions, a cell density of OD₆₀₀
~50 (dry cell weight ~13 g/L) with >99% cell viability was achieved
in the casamino acids supplemented buffered-minimal-media in 300 to
1000 μl culture volume/well. We have devised a simplified method
for coating of the culture supernatant on the polystyrene surface for
immunoassay. Clones for secretory expression of envelope domain III of
dengue virus serotype-1 under the control of inducible and constitutive
promoter were screened using the developed method. Described microscale
cultivation strategy can be used for rapid high-throughput screening of P. pastoris
clones, media optimization, and high-throughput recombinant protein
production. The knowledge gained through this work may also be applied,
to other suspension cultures, with some modifications.