The Effect of Limiting Phosphate Using the HMS174 Cell Line - Surprising results from a case study using the HMS174 cell line. - BioPharm International

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The Effect of Limiting Phosphate Using the HMS174 Cell Line
Surprising results from a case study using the HMS174 cell line.


BioPharm International
Volume 23, Issue 7

Up-Regulation of Alkyl hydroxide Reductase Subunit C in Culture Without Phosphate Feed


Figure 3
During culture growth, samples were taken for media and cell pellet analysis. The pellets isolated after induction showed an increase in the production of a 22 kD protein in the culture without phosphate feed compared with the culture with added phosphate feed (Figure 3). This 22 kD protein was found to have strong sequence homology to alkyl hydroperoxide reductase (subunit C), an oxygen radical scavenger.9 The presence of such an oxygen radical scavenger indicates the highly oxidative state of the HMS174 culture fed only glucose. It is assumed that without additional phosphate, glycolysis is somewhat inhibited. With lower amounts of pyruvate being formed, converted to acetyl-CoA and metabolized in the citric acid cycle, the synthesis of NADH and FADH2 is compromised and thus inhibits the oxidative phosphorylation pathway. This would indeed be the case if the culture fed only glucose switched to the glyoxylate pathway that yields only 1 NADH molecule per acetate molecule.

Equivalence of Recombinant Protein Production on the Two Cultures


Table 1. Ratio of wet cell weight and inclusion body weight. The HMS174 culture with and without phosphate added to the glucose feed produced the same amount of inclusion body protein during a 2 h induction with IPTG.
The production of recombinant protein product per gram wet cell paste is identical for the two cultures. As stated earlier, the growth rate of the HMS174 culture was substantially reduced when fed glucose only, compared to the culture being fed glucose and phosphate. However, this did not translate into a reduction of product formation per gram of cell paste when the cultures were induced with IPTG. The amount of product formation was determined after lysing the cell culture and isolating the inclusion bodies through centrifugation (Table 1). The expression of the recombinant protein in both cultures also was equivalent on SDS-PAGE stained with Coomassie blue (Figure 3). The amount loaded per lane was based on OD equivalents.

DISCUSSION

Recombinant E. coli host strains such as K or B vary in their ability to produce sufficient quantities of recombinant proteins, to grow to high cell densities on different media, and most importantly, to show a robustness for changing metabolic demands throughout the fermentation process. Although the B strains produce less acetate because of an activated glyoxylate shunt,10 the host K strain HMS174 (DE3) containing the pET29a plasmid and gene of interest has not shown any negative effects from the production of acetate during high growth rates. During the exponential growth phase, the cultures fed glucose plus phosphate or glucose only cultures reached a maximum growth rate of 2.7 h and 1.5 h, respectively. Residual phosphate levels for both cultures decreased soon after the feed was started with the culture without phosphate feed reaching undetectable levels at the 3 h time point (Figure 2B). Phosphate in the culture with added phosphate feed was detectable throughout the growth, although decreasing steadily.

After induction with IPTG, not surprisingly, the culture with added phosphate feed produced twice the amount of recombinant product as the culture without phosphate feed because of the increased cell density. What was surprising was that both cultures produced identical amounts of recombinant product per g of wet cell paste. This was presented as a ratio of g of inclusion body per g of wet cell paste recovered. The equal production was confirmed by SDS-PAGE. For this to take place, the culture without phosphate feed must make use of other carbon sources besides glucose. Substances that contain 2 carbons, such as acetate, are an obvious choice. Post induction, the differently fed cultures showed markedly different residual profiles for glucose, acetate, and phosphate. Ironically, the culture with added phosphate feed immediately began uptake of acetate, glucose, and phosphate, whereas the culture fed only glucose maintained acetate levels and continued to increase residual glucose levels (Figure 2C).

The immediate uptake of acetate in the culture with added phosphate, after induction, suggests that this cell culture is already primed for the metabolism of a 2-carbon source, such as acetate. This contradicts the general assumption that when E. coli is grown on a carbon source such as glucose, the glyoxylate bypass is unnecessary and is shut down by the dephosphorylation of the enzyme isocitrate dehydrogenase.11 This allows isocitrate to be forwarded through the tricarboxylic acid cycle. Moreover, when considering the two different post-induction metabolic states of these cultures, there is no noticeable reduction of product formation in the culture fed only glucose of HMS174. The reasons for this are not clear.

In further studies on the HMS174 host cell strain, the immediate use of the residual acetate after induction could indicate the possibility of a new mixed feed strategy using glucose or phosphate before induction, then adding acetate separately during induction. This may increase the recombinant protein product formation and further elucidate the metabolic pathways used in an HMS174 induced culture.


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