Expression of human T-cell leukemia virus type I protease in Escherichia coli.
Human T-cell leukemia virus type I (HTLV-I) genome is believed to encode its own protease, although the protease has not yet been detected. To identify the HTLV-I protease, an in-frame gag (3' portion)-prt region was expressed in Escherichia coli. The 14-kDa product was detected using antisera against a synthetic peptide mimicking the fragment of HTLV-I protease, although the molecular weight of the primary translational product was 27,000. A cell extract had a proteolytic activity to cleave a synthetic peptide substrate containing the cleavage site of gag p19/p24 at the correct site in vitro. Replacement of the putative active site Asp-64 with Gly abolished both in vivo processing activity and in vitro proteolytic activity. These results suggest that the 14-kDa product is the mature enzymatically active HTLV-I protease generated through posttranslational autoprocessing in E. coli.
Hayakawa
T
T
Biotechnology Research Laboratories, Takeda Chemical Industries, Ltd., Osaka, Japan.
Misumi
Y
Y
Kobayashi
M
M
Ohi
Y
Y
Fujisawa
Y
Y
Kakinuma
A
A
Hatanaka
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005814:Genes, Viral; D015750:Genes, gag; D016679:Genome, Viral; D015368:Human T-lymphotropic virus 1 / Q000201:enzymology / Q000235:genetics*; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D016366:Open Reading Frames; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D010455:Peptides; D011994:Recombinant Proteins; D010450:Endopeptidases