Antifoam Addition to Shake Flask Cultures of Recombinant Pichia Pastoris Increases Yield - Essay Example

Invariably used without prior consideration on their potential impacts on yeast cells, protein product, or protein purification, these instruments require substantial study to achieve optimum output in recombinant protein production with P.  pastoris.

We want to find out if antifoams (Antifoam A (Sigma), Antifoam C (Sigma), J673A (Struktol), P2000 (Fluka), and SB2121 (Struktol)) affect recombinant yield (recombinant green fluorescent protein (GFP)) in shake-flask of P. pastoris and investigate their essential mechanisms.

To specifically find potential results regarding the above concern, we ensure uniform employment of P. pastoris strain X33 throughout the entire experimental procedure. Cells were cultured in shake flasks and tested with antifoams to find their respective and remarkable connection with recombinant protein yield.

Materials and Methods

Yeast strains and culturing condition

Pichia pastoris cultured in shake flask in medium (pH 6.0, 1 M potassium phosphate buffer, 300 C and 220 rpm).

Initial biomass accumulation stage (BMGY medium – 1% yeast extract, 2% peptone, 100mM potassium phosphate pH 6.0, 1.34% YNB, 4x10-5 % biotin, 1% glycerol)

Induce production of GFP (BMMY medium – 1% yeast extract, 2% peptone, 100mM potassium phosphate pH 6.0, 1.34% YNB, 4x10-5 % biotin, 0.5% methanol)

Shake flask cultures

Cell culture & accumulation of biomass (50 ml BMGY in 250 ml baffled shake flasks)

Inoculation (OD595 1.0, 100 ml non-baffled shake flasks)

Antifoam evaluation (triple replicates in the following concentrations: 0%, 0.2%, 0.4%, 0.6%, 0.8%, or 1.0% (v/v))

Antifoam agents (autoclaved)

Schill and Schelinger’s Struktol SB2121 (polyakylene glycol)

Schill and Schelinger’s Struktol J673A (alkoxylated fatty acid ester on a vegetable base)

Fluka P2000 (polypropylene glycol)

Sigma Antifoam A (30% emulsion of silicone polymer)

Sigma Antifoam C (30% emulsion of silicone polymer)

Fluorescence measurements

One-way ANOVA for finding a significant difference between any means

Total protein analysis

One-way ANOVA test and Dunnett’s multiple comparison tests

Bartsch test

Protocol outline from Denkov and colleagues

KLa determination

Dissolved oxygen measurements

DO fluorescent sensors (PreSens)

Flow cytometry

One-way ANOVA test and Dunnett’s multiple comparison tests

Results

Antifoam addition affects total GFP yield in shake flasks

A remarkable trend of GFP yield increase was observed when antifoams were incorporated in shake flasks. The concentration was somewhere in the range of 0.4%-1% v/v. Highest yield achieved at the addition of 1% P2000, 1% SB2121, 1% J673A, 0.6% Antifoam A, and 0.8% Antifoam C.

Antifoam addition does not affect cell viability

There were no dead cells present in cultures containing 0% and 0.6% Antifoam respectively.

Antifoams affect foam volume

As determined by the Bartsch test, those antifoams employed in Figure 1 were tested for their destruction capacity. All antifoams in general were effective at foam destruction. J673A was the most effective at foam destruction and best at increasing GFP yield, while Antifoam C was less destructive.

kLa and the total yield

In general, there was no correlation between kLa and total yield for any of the conditions tested in the study, and density of the cultures, which only implies that kLa may not directly contribute to the increased protein yield of antifoams.

DO and antifoams: the case of P2000 and SB2121

After 12 hours, the DO in the flasks significantly showed no difference in the flasks with or without antifoams. This in general implies that the level of DO content of cultures containing antifoam is proof that antifoams do not have any direct contributing factor to affect the DO level.

Antifoam and retained and secreted GFP

Antifoams do not influence the growth of the cells, but there was an enhancement in the level of amount secreted into the medium. Using one-way ANOVA and Dunnett’s multiple comparisons tests the statistical significance of this result can be evaluated.

Conclusion

In general and based on the above results, we have potentially shown that antifoaming agents on shake flask cultures of P. pastoris increase the yield of the production of recombinant protein. One potential benefit of our results is about paving the way for understanding how antifoams could be associated with microbial cell factories based on their probable interactions. Based on their respective mechanisms, we also found that one group of antifoams could increase the total amount of produced protein and secreted per cell (Antifoam A, Antifoam C, J673A). The other group could also generate high yield by increasing culture’s density (P2000, SB2121).