Characterization of up-regulated proteases in an industrial recombinant Escherichia coli fermentation (original) (raw)

Proteolytic degradation of recombinant proteins is an industry -wide challenge in host organisms such as Escherichia coli. These proteases have been linked to stresses, such as the stringent and heat -shock responses. This study reports the dramatic up -regulation of protease activity in an industrial recombinant E. coli fermentation upon induction. The objective of this project was to detect and characterize up -regulated proteases due to recombinant AXOKINE 1 1 1 1 overexpression upon IPTG induction. AXOKINE 1 1 1 1 is a 22 -kDa protein currently in clinical trials as a therapeutic for obesity associated with diabetes. AXOKINE 1 1 1 1 was expressed in both the soluble and inclusion body fractions in E. coli. Sodium dodecyl sulfate gelatin -polyacrylamide gel electrophoresis ( SDS -GPAGE ) was used to analyze the up -regulated protease activity. Western blot analysis showed degraded AXOKINE 1 1 1 1 in both the soluble and insoluble fractions. Protease inhibitors were used to characterize the proteases. The proteases were ethylenediaminetetraacetic acid ( EDTA ) sensitive. The protease activity increased in the presence of phenyl -methyl sulfonyl -fluoride ( PMSF ), a serine protease inhibitor. The incubation buffer composition was varied with respect to Mg 2 + and ATP, and the protease activity was ATP independent and Mg 2 + dependent. A two -dimensional electrophoresis technique was used to estimate the pI of the proteases to be between 2.9 and 4.0.