Characterization of up-regulated proteases in an industrial recombinant Escherichia coli fermentation.
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 overexpression upon IPTG induction. AXOKINE is a 22-kDa protein currently in clinical trials as a therapeutic for obesity associated with diabetes. AXOKINE 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 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 Mg2+ and ATP, and the protease activity was ATP independent and Mg2+ dependent. A two-dimensional electrophoresis technique was used to estimate the pI of the proteases to be between 2.9 and 4.0.
Jordan
G L
GL
Department of Chemical Engineering, New Mexico State University, Las Cruces 88003, USA.
Harcum
S W
SW
D000255:Adenosine Triphosphate / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D005285:Fermentation; D008274:Magnesium / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D015854:Up-Regulation
D011480:Protease Inhibitors; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D010450:Endopeptidases; D008274:Magnesium