Refolding of Npro fusion proteins.
The autoprotease Npro significantly enhances expression of fused peptides and proteins and drives the formation of inclusion bodies during protein expression. Upon refolding, the autoprotease becomes active and cleaves itself specifically at its own C-terminus releasing the target protein with its authentic N-terminus. Npro wild-type and its mutant EDDIE, respectively, were fused N-terminally to the model proteins green fluorescent protein, staphylococcus Protein A domain D, inhibitory peptide of senescence-evasion-factor, and the short 16 amino acid peptide pep6His. In comparison with the Npro wild-type, the tailored mutant EDDIE displayed an increased rate constant for refolding and cleavage from 1.3 x 10(-4) s(-1) to 3.5 x 10(-4) s(-1), and allowed a 15-fold higher protein concentration of 1.1 mg/mL when studying pep6His as a fusion partner. For green fluorescent protein, the rate constant was increased from 2.4 x 10(-5) s(-1) to 1.1 x 10(-4) s(-1) when fused to EDDIE. When fused to small target peptides, refolding and cleavage yields were independent of initial protein concentration, even at high concentrations of 3.9 mg/mL, although cleavage rates were strongly influenced by the fusion partner. This behavior differed from conventional 1st order refolding kinetics, where yield strongly depends on initial protein concentration due to an aggregation reaction of higher order. Refolding and cleavage of EDDIE fusion proteins follow a monomolecular reaction for the autoproteolytic cleavage over a wide concentration range. At high protein concentrations, deviations from the model assumptions were observed and thus smaller rate constants were required to approximate the data.
Kaar
Waltraud
W
Austrian Center of Biopharmaceutical Technology, Vienna, Austria.
Ahrer
Karin
K
Dürauer
Astrid
A
Greinstetter
Sabine
S
Sprinzl
Wolfgang
W
Wechner
Philipp
P
Clementschitsch
Franz
F
Bayer
Karl
K
Achmüller
Clemens
C
Auer
Bernhard
B
Hahn
Rainer
R
Jungbauer
Alois
A
D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D010450:Endopeptidases; C505229:N(pro) protein, swine fever virus