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

• Published in: Applied and Environmental Microbiology 2005-04, Vol.71 (4), p.1717-1728
• Main Authors: Aldor, Ilana S, Krawitz, Denise C, Forrest, William, Chen, Christina, Nishihara, Julie C, Joly, John C, Champion, Kathleen M
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - metabolism; Biological and medical sciences; Biotechnology; CD18 Antigens - immunology; Electrophoresis, Gel, Two-Dimensional; Escherichia coli; Escherichia coli - growth & development; Escherichia coli - metabolism; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Fermentation; Fundamental and applied biological sciences. Psychology; Gene expression; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Health. Pharmaceutical industry; Heat-Shock Proteins - metabolism; Image Processing, Computer-Assisted; Immunoglobulin Fragments - biosynthesis; Immunoglobulin Fragments - genetics; Industrial applications and implications. Economical aspects; Industrial Microbiology - methods; Microbiology; Other active biomolecules; Physiology and Biotechnology; Production of active biomolecules; Proteome; Quantitative genetics; Recombinant Proteins - genetics; Recombinant Proteins - metabolism
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/AEM.71.4.1717-1728.2005

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.