Production of chemokines CTAPIII and NAP/2 by digestion of recombinant ubiquitin-CTAPIII with yeast ubiquitin C-terminal hydrolase and human immunodeficiency virus protease.
Recombinant yeast ubiquitin C-terminal hydrolase (YUH1), which has an N-terminal (His)(6) tag, 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: [see text] YUH1 cleaved at the peptide bond formed by the C-terminal Gly(76) of ubiquitin (Ub) and the N-terminal Asn(1) of the 85-residue peptide CTAPIII. The HIV-1 Pr cleaved between Tyr(15) and Ala(16), 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.
Mildner
A M
AM
Protein Science, Cell and Molecular Biology, Pharmacia and Upjohn, 301 Henrietta Street, Kalamazoo, Michigan 49007, USA.
Paddock
D J
DJ
LeCureux
L W
LW
Leone
J W
JW
Anderson
D C
DC
Tomasselli
A G
AG
Heinrikson
R L
RL
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D016333:HIV Protease / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014452:Ubiquitins / Q000378:metabolism; D001620:beta-Thromboglobulin
D017931:DNA Primers; C061615:PPBP protein, human; D010455:Peptides; D011994:Recombinant Proteins; D014452:Ubiquitins; D001620:beta-Thromboglobulin; C009862:connective tissue-activating peptide; D010450:Endopeptidases; D016333:HIV Protease; C059237:ubiquitin-Nalpha-protein hydrolase