| Abstrakt: |
Recombinant yeast ubiquitin C-terminal hydrolase (YUH1), which has an N-terminal (His)6tag, and an autolysis-resistant mutant of the human immunodeficiency virus-1 protease (HIV-1 Pr) have been used as specific proteases to yield peptides from a ubiquitin conjugate. In the present example, connective tissue-activating peptide (CTAPIII) and neutrophil-activating peptide 2 (NAP/2) were generated by digestion of a ubiquitin–CTAPIII conjugate with YUH1 and HIV Pr, respectively, as indicated below: YUH1 cleaved at the peptide bond formed by the C-terminal Gly76of ubiquitin (Ub) and the N-terminal Asn1of the 85-residue peptide CTAPIII. The HIV-1 Pr cleaved between Tyr15and Ala16, the N-terminal Ala of the 70-residue peptide NAP/2. Both enzymes produced authentic peptides from the Ub fusion protein, with a nearly 100% yield. The liberated CTAPIII and NAP/2 were separated from (His)6–Ub, the trace amounts of unreacted (His)6–Ub–CTAPIII, HIV-1 Pr, and the (His)6–YUH1 by passage over a nickel-chelate column; the final yield was about 10 mg of peptide/liter of cell culture. (His)6–YUH1, the HIV Pr mutant, and the (His)6–Ub–CTAPIII substrate were all expressed individually in Escherichia coli.(His)6–YUH1 and (His)6–Ub–CTAPIII were highly expressed in a soluble form, but about 75% of the total (His)6–YUH1 was also found in inclusion bodies. Both proteins from the soluble fractions were easily purified in a single step by immobilized metal ion affinity chromatography with a yield of about 27 mg of (His)6–Ub–CTAPIII and 13.6 mg of (His)6–YUH1 protein/liter of cell culture. Chemotactic factor activity, as assessed by the neutrophil shape change assay, was observed for NAP/2, but not for CTAPIII. This strategy, which employs YUH1 and the HIV-1 Pr as tools for the highly selective cleavage of the chimeric substrate, should be applicable to the large-scale production of a variety of peptides. |
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