Characterization and application of a periplasmic protein releasing system for extracellular recombinant protein production in Escherichia coli

[摘要] Escherichia coli is commonly used as a host in the production of high value therapeutic proteins. However, one major drawback associated with E. coli being a host is the intracellular location of product proteins. As a result, mechanical cell disruption is commonly employed in biotechnology industry for protein release. Unfortunately, mechanical disintegration always contaminates the target protein with other cellular substances. On the other hand, progress in the study of protein translocation across membrane often allows the transport of proteins from the cytoplasm to the periplasm. However, the outer membrane still presents a barrier for their extracellular release. The objective of this work is to bridge the gap between protein translocation and extracellular protein release. The main focus is to characterize a periplasmic protein release system and to apply the system to different processes for extracellular protein production. Bacteriocin Release Protein (BRP) and glycine were the two systems studied using a-amylase as the model protein. Both agents were shown to be effective in periplasmic protein release. When used alone in a batch fermentor, BRP released 41% of the periplasmic a-amylase extracellularly without causing substantial cell lysis. Similarly, glycine achieved a 45% protein release. Synergistic combination of BRP and glycine resulted in further increase in a-amylase release. At an optimal combination, 78% of a-amylase was released. Application of the release system for continuous extracellular protein production was successful. A maximum productivity of 8.3 units/ml/hr was obtained, of which one third was in the extracellular broth. Mathematical simulation suggested that higher expression levels of a-amylase at slower growth rates is mainly due to a higher plasmid copy number. Incorporation of hollow fiber cartridges into a chemostat process allowed continuous production of cell-free proteins as well as retention of recombinant cells. At an optimal dilution rate, 42% of a-amylase was produced in a cell-free form. Substitution of the Amp$sp{m r}$ gene in pBR322-amy with a Km$sp{m r}$ gene in an attempt to improve the performance of the release system resulted in a higher accumulation of a-amylase in the cytoplasm. The observation demonstrated that antibiotic markers are important parameters in a-amylase translocation and distribution.

[发布机构] Rice University

[效力级别] Molecular biology