Human T-cell leukemia virus type 1 protease protein expressed in Escherichia coli possesses aspartic proteinase activity.
We amplified the human T-cell leukemia virus type 1 (HTLV-1) protease gene fragment by polymerase chain reaction (PCR) and cloned it into a pUC plasmid vector. DNA sequencing data of the protease gene fragment indicated that it contained an open reading frame capable of encoding the active HTLV-1 protease. To express a fusion protein of beta-galactosidase linked with the HTLV-1 protease in Escherichia coli, a plasmid DNA was constructed by inserting the HTLV-1 protease gene DNA into a procaryotic expression vector, pUEX2, consisting of a lacZ gene directed by a lambda phage Pr promoter and designated pUEX-pro. By Western blot analysis using anti-beta-galactosidase antibody, a bigger molecular size band than that of the control beta-galactosidase molecule was observed in E. coli cells transformed with pUEX-pro but not with control pUEX2, suggesting that the particular fusion protein was successfully expressed. This recombinant protease protein in the E. coli cell lysate was demonstrated to be able to cleave the decapeptide substrates composed of amino acid sequences containing proteolytic cleavage sites in the HTLV-1 gag precursor polyprotein. The gag precursor polyprotein expressed in the mammalian cells by the recombinant vaccinia virus system was also expectedly cleaved by this enzyme. Significant inhibition of this protease activity by pepstatin A, an aspartic proteinase-specific inhibitor, confirms that HTLV-1 protease is a member of the aspartic proteinase group as suggested previously. Since the crude lysate without purification is utilized sufficiently as a native HTLV-1 protease reagent, this protease preparation is easily applicable to the large scale screening of HTLV-1 protease inhibitors for the treatment of diseases caused by HTLV-1.
Saiga
A
A
Shionogi Institute for Medical Science, Osaka, Japan.
Tanaka
T
T
Orita
S
S
Sato
A
A
Sato
S
S
Hachisu
T
T
Abe
K
K
Kimura
Y
Y
Kondo
Y
Y
Fujiwara
T
T
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000032:analysis; D010450:Endopeptidases / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D015683:Gene Products, gag / Q000378:metabolism; D015368:Human T-lymphotropic virus 1 / Q000201:enzymology*; D008969:Molecular Sequence Data; D010436:Pepstatins / Q000494:pharmacology; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014170:Transformation, Genetic; D001616:beta-Galactosidase / Q000378:metabolism
D004279:DNA, Viral; D015683:Gene Products, gag; D010436:Pepstatins; D011993:Recombinant Fusion Proteins; C014195:Streptomyces pepsin inhibitor; D001616:beta-Galactosidase; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C079711:HTLV-1 protease; C031375:pepstatin