Growth Kinetics of Escherichia coli and Expression of a Recombinant Protein and Its Isoforms under Heat Shock Conditions

Abstract

Preinduction culture conditions were found to have significant impact on the expression and post-translational modification of a recombinant human protein in Escherichia coli under heat shock conditions (30 to 42 degrees C shift). Higher preinduction growth rates (micrograms) favored better cell viability, greater cell mass yields, and increased cloned gene expression during induction. Formation of recombinant protein isoforms (those containing N epsilon-modified lysine residues) exhibited an increasing trend with increasing micrograms. The different extents of post-translational modifications were suspected to be linked to the different concentrations of certain heat shock protein chaperones resulting from different micrograms. In view of the extensive involvement of E. coli heat shock proteins in cellular activities-including the synthesis, processing, modification, and degradation of proteins-at elevated temperatures, it is believed that micrograms dictated the cellular resources available for synthesizing the heat shock proteins required for cell survival, which in turn determined the ability of the cells to respond to the heat shock. With a higher micrograms both the synthesis of host proteins (as indicated by cell growth and survival) and the cloned gene expression were enhanced. The results demonstrate that there exists an intermediate micrograms for optimum production of the unmodified foreign protein in a heat shock environment. More importantly, they also illustrate the feasibility of improving the recombinant protein homogeneity in fermentation, thereby facilitating downstream processing.

Cell engineering of Escherichia coli allows high cell density accumulation without fed-batch process control

“…This was earlier found also for other proteins and other expression systems [3,20]. However, expression of a recombinant protein at high growth rate was also reported to increase the production of a modified protein product [21]. At a growth rate of 0.2 h -1 , about 45% of the product was correctly formed and this amount was decreased to 30% when the growth rate increased to 0.5 h -1 .…”

Limiting factors in Escherichia coli fed‐batch production of recombinant proteins

“…Very few reports are at present available on the effect of the feed profile on the quality of the product. One example is given by Ryan et al (1996), who showed that, for an unnamed product, a high productivity was inversely proportional to a high product quality and more isoforms of the protein were present the higher the production rate. Also, Hellmuth et al (1994) showed that there is an optimum in the achieved aprotinin::␤-galactosidase fusion protein activity as a function of the specific growth rate.…”

Microbial production of virus-like particle vaccine protein at gram-per-litre levels

“…Pre-induction culture conditions, generally the specific growth rate, significantly affect the specific productivity and specific production rate of heterologous protein expression in recombinant E. coli (Lim and Jung, 1998;Ryan et al, 1996). The specific growth rate is maximised during batch phase and decreases gradually with time in the fed-batch phase.…”