Proteomic Profiling of Recombinant Escherichia coli in High-Cell-Density Fermentations for Improved Production of an Antibody Fragment Biopharmaceutical

Abstract

By using two-dimensional polyacrylamide gel electrophoresis, a proteomic analysis over time was conducted with high-cell-density, industrial, phosphate-limited Escherichia coli fermentations at the 10-liter scale. During production, a recombinant, humanized antibody fragment was secreted and assembled in a soluble form in the periplasm. E. coli protein changes associated with culture conditions were distinguished from protein changes associated with heterologous protein expression. Protein spots were monitored quantitatively and qualitatively. Differentially expressed proteins were quantitatively assessed by using a t-test method with a 1% false discovery rate as a significance criterion. As determined by this criterion, 81 protein spots changed significantly between 14 and 72 h (final time) of the control fermentations (vector only). Qualitative (on-off) comparisons indicated that 20 more protein spots were present only at 14 or 72 h in the control fermentations. These changes reflected physiological responses to the culture conditions. In control and production fermentations at 72 h, 25 protein spots were significantly differentially expressed. In addition, 19 protein spots were present only in control or production fermentations at this time. The quantitative and qualitative changes were attributable to overexpression of recombinant protein. The physiological changes observed during the fermentations included the up-regulation of phosphate starvation proteins and the down-regulation of ribosomal proteins and nucleotide biosynthesis proteins. Synthesis of the stress protein phage shock protein A (PspA) was strongly correlated with synthesis of a recombinant product. This suggested that manipulation of PspA levels might improve the soluble recombinant protein yield in the periplasm for this bioprocess. Indeed, controlled coexpression of PspA during production led to a moderate, but statistically significant, improvement in the yield.

TheEscherichia coliProteome: Past, Present, and Future Prospects

“…Indeed, the heat-inducible and inorganic phosphorus-responsive promoters have been widely used in numerous laboratories (3,37,38,115,139). In addition, the genes encoding proteins that confer tolerances to acid, heat, and toxic substances have been successfully used for the improvement of cellular properties and enhanced degradation of toxic chemicals.…”

“…Proteomic analysis has been used to directly monitor cellular changes occurring during the production of heterologous proteins in E. coli and develop efficient strains for the enhanced production of bioproducts (3,37,38,97,98,114,115,116,135,138,160,162,235,306) and biodegradable polymers (99,139). Furthermore, many of these proteomic studies have been performed in large-scale processes employing E. coli and recombinant E. coli for industrial applications (3,37,38,97,114,115,116,135,138,139,145,241,245,325). In addition, proteomic studies for analyzing the composition of inclusion bodies (IBs) (98,101,135,138,246,247) have been carried out in order to improve the quality (or uniformity) of the desired product and the downstream process of recombinant proteins such as protein purification and refolding.…”

Towards systems metabolic engineering of microorganisms for amino acid production

“…Proteome analysis is also a powerful tool for systems-level analysis of whole cell proteins, even though it does not provide a true global picture owing to unknown proteins. It can be used to identify genes to be engineered to develop superior strains [29,30]. Proteome reference maps, which enable identification of proteins on 2D gel, are used for comparative proteome profiling [31][32][33][34].…”

High-level production of a single chain antibody against anthrax toxin in Escherichia coli by high cell density cultivation

“…In E. coli, large-scale production of recombinant proteins including various antibodies has been successfully accomplished by high cell density cultivation (HCDC) technologies that can achieve cell densities higher than OD 600 of 100 [11,12]. Since Carter et al [13] first reported the production of 1-2 g/L of humanized F(ab) 2 in a 10 L culture of E. coli, HCDC of E. coli has been used for the production of many antibody fragments as well as full length IgG [14][15][16][17][18][19][20]. Horn et al [17] performed the fed-batch fermentation for the production of dimeric miniantibodies against EGF-receptor in 10 L bioreactor, and they could successfully produce more than 3 g/L of soluble antibodies, which is the best reported so far.…”