Expression and refolding of mite allergen pro-Der f1 from inclusion bodies in Escherichia coli (1)
pro-Der f1 as a template and synthetic oligonucleotides 5′-GGAATTCCATATGCGTCCAGCTTCAATCAAAAC-3′ and 5′-CCGCTCGAGTTATTACTTTTCGAACTGCGGGTGGCTCCACATGATTACAACGTATGGATA-3′ as primers, and inserted at the Nde I/Xho I site of pET-44a plasmid.
Protein was produced in the form of an inclusion body. Pro-rDer f1 was identified by Western blot and showed a single band, indicating that pro-rDer f1 was successfully expressed with the Strep II tag, which was located at the C-terminal end of the expressed protein.
Refolding efficiency between the dilution and SEC method was somewhat different. The protease activity increased with increasing concentrations of GSH and GSSG but was slightly decreased when the concentrations were above 3 mM/0.3 mM GSSG. Slower mobile phase flow rates allow pro-rDer f1 to stay inside of the column longer and results in increased enzyme activity recovery.
Pro-rDer f1 was separated into two peaks, peaks A and B. The leading peak A is aggregated protein, peak B is refolded protein, and peak C is DTT and denaturants.
SEC refolded pro-rDer f1 into a bioactive protein more effectively than dilution.
Reduced and oxidized glutathione (GSH/GSSG) are the most commonly used oxido-shuffling systems. The molar ratios of reduced to oxidized glutathione recom- mended for papain-like cysteine proteases is 10:1. When the ratio of GSH to GSSG was 10:1, the most effective concentration for protein refolding of pro-rDer f1 was 3 mM GSH and 0.3 mM GSSG. Hence, removal of the signal peptide is a helpful choice when the protein is expressed in E. coli. In any case, the pro-peptide regulates enzymatic activity and may function as an intramolecular chaperone in the folding process. Therefore, when rDer f1 is directly expressed in mature form in E. coli, the inclusion bodies do not refold correctly and show very low IgE binding activities. IgE binding activity to the mite group 1 allergens (Der f1 and Der p1) is highly dependent on the tertiary structure of the allergens.