Recombinant Escherichia coli cultures are used to manufacture numerous therapeutic proteins and industrial enzymes, where many of these processes use elevated temperatures to induce recombinant protein production. The heat-shock response in wild-type E. coli has been well studied. In this study, the transcriptome profiles of recombinant E. coli subjected to a heat-shock and to a dual heat-shock recombinant protein induction were examined. Most classical heat-shock protein genes were identified as regulated in both conditions. The major transcriptome differences between the recombinant and reported wild-type cultures were heavily populated by hypothetical and putative genes, which indicates recombinant cultures utilize many unique genes to respond to a heat-shock. Comparison of the dual stressed culture data with literature recombinant protein induced culture data revealed numerous differences. The dual stressed response encompassed three major response patterns: induced-like, in-between, and greater than either individual stress response. Also, there were no genes that only responded to the dual stress. The most interesting difference between the dual stressed and induced cultures was the amino acid-tRNA gene levels. The amino acid-tRNA genes were elevated for the dual cultures compared to the induced cultures. Since, tRNAs facilitate protein synthesis via translation, this observed increase in amino acid-tRNA transcriptome levels, in concert with elevated heat-shock chaperones, might account for improved productivities often observed for thermo-inducible systems. Most importantly, the response of the recombinant cultures to a heat-shock was more profound than wild-type cultures, and further, the response to recombinant protein induction was not a simple additive response of the individual stresses.

Culture conditions that affect product quality are important to the successful operation and optimization of bioreactors. Previous studies have demonstrated that enzymes, such as proteases and sialidases, accumulate in batch bioreactors. These enzymes are known to be detrimental to the quality of recombinant glycoproteins. Bioreactor temperature has been used to control cell growth and recombinant protein production rates. However, the effect of culture temperature on the production of proteases and sialidases has not been investigated. In this study, Chinese hamster ovary cells were cultured with a temperature profile that decreased from 37 to 34 °C over 8 days and with a constant temperature of 37 °C. Analysis of extracellular protease activity indicated that two major proteases were present (50 and 69 kDa). The 50 kDa protease activity decreased similarly with time for both culture conditions. The 69 kDa protease activity increased with time for both culture conditions. The constant-temperature cultures had significantly lower 69 kDa protease levels compared to the ramped- temperature cultures in the early stationary phase. Intracellular sialidase activity was present in both cultures. The intracellular sialidase activity increased dramatically for both culture conditions immediately after the cells were inoculated into fresh medium. The initial peak in intracellular sialidase activity was followed by a first- order decay. The intracellular sialidase activities for the two culture conditions were not significantly different. The production of recombinant tissue type plasminogen activator was not significantly different for the two culture conditions. Thus, the previously hypothesized advantages that lower culture temperatures have reduced protease activity and improved productivity do not appear to be universal.