A novel outer-membrane-associated protease in Escherichia coli

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Recombinant human gamma interferon produced in Escherichia coli is susceptible to degradation by endogenous proteases.
• Proteolytic activity in E. coli can impact the yield and integrity of recombinant proteins.

Purpose of the Study:

• To identify and characterize the endogenous protease responsible for degrading human gamma interferon in E. coli.
• To determine the substrate specificity and inhibitory properties of this novel protease.

Main Methods:

• Cell disruption of recombinant E. coli producing human gamma interferon.
• Analysis of degradation products using molecular weight determination.
• Assay of proteolytic activity associated with the E. coli outer membrane.
• Testing the effects of various protease inhibitors and metal ions.
• Substrate cleavage analysis using Dynorphin A(1-13).

Main Results:

• Human gamma interferon was cleaved at Lys-131-Arg-132 and Arg-142-Arg-143, yielding 17,500 and 16,000 MW products.
• Proteolytic activity was localized to the E. coli outer membrane.
• The enzyme was insensitive to common protease inhibitors but inhibited by benzamidine, Zn2+, and Cu2+.
• Dynorphin A(1-13) was preferentially cleaved at Arg-6-Arg-7, indicating specificity for consecutive basic residues.

Conclusions:

• A new E. coli outer membrane protease, designated protease VII, specifically cleaves peptide bonds between consecutive basic amino acids.
• Protease VII differs from previously identified membrane proteases (IV, V, and VI) in its cleavage specificity.
• Understanding protease VII activity is crucial for optimizing recombinant protein production in E. coli.