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EDDIE fusion proteins: Triggering autoproteolytic cleavage

Paper ID Volume ID Publish Year Pages File Format Full-Text
35090 45074 2009 8 PDF Available
EDDIE fusion proteins: Triggering autoproteolytic cleavage

Heterologous proteins are often poorly expressed in Escherichia coli and especially small peptides are prone to degradation. Npro autoprotease fusion proteins, deposited as inclusion bodies in E. coli, are a versatile tool for peptide and protein overexpression and generate an authentic N terminus at the target molecule. Autoproteolytic cleavage and subsequent release of the fusion partner are initiated upon refolding. Fusion proteins with the Npro mutant EDDIE follow a monomolecular reaction. The reaction rate was only dependent on chaotrope concentration, decreasing exponentially by a factor of 1.2–1.5 for urea and by a factor of 2.1–5.3 for GuHCl. The first amino acid of the target peptide had a major impact on the reaction rate studying a set of model peptides. Reaction rates were in the range of 2.2 × 10−4 to 7.3 × 10−5 s−1 and could be increased up to fivefold by exchanging the first amino acid of the target peptide. A panel of biophysical methods was used to assess EDDIE secondary and tertiary structure. Immediate formation of secondary structure and slight increase in β-sheet content of approximately 5% over the course of the cleavage reaction was observed and interpreted as aggregation. Aggregation and cleavage occurred simultaneously. EDDIE has a relatively loose structure with the cleavage site exhibiting the lowest solvent exposure. We hypothesize that this is the mechanism for establishing a spatial proximity between cleavage site and the catalytic centre of the autoprotease. Fluorescence measurements revealed that further structural changes did not occur after the initial hydrophobic collapse. Thus, the overall reaction is predominantly controlled by cleavage kinetics and refolding kinetics does not play a major role.

ACN, acetonitrile; ATR-FTIR, attenuated total reflectance Fourier transform infrared; b, k0 associated parameter; c, chaotrope concentration; R2, coefficient of determination; conc., concentration; C (Cys), cysteine; GuHCl, guanidine hydrochloride; 6His,
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EDDIE fusion proteins: Triggering autoproteolytic cleavage
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 44, Issue 11, November 2009, Pages 1217–1224
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Physical Sciences and Engineering Chemical Engineering Bioengineering