Expression and characterization of the reverse transcriptase enzyme from type 1 human immunodeficiency virus using different baculoviral vector systems.
To produce the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) in amounts required to study its structure and function, the p66 enzyme subunit was expressed using two different baculovirus vectors in Sf158 insect host cells. Both vectors permitted an efficient HIV-1 RT expression. The resulting products were purified up to 90% homogeneity, characterized, and investigated for their susceptibility to digestion with various proteases. The recombinant baculoviral RT obtained with the pAc373 expression vector was purified as a p66/p60 heterodimer. The recombinant His-RT was expressed with the pBlueBacHis vector. Thereby, the protein was tagged with an N-terminal hexahistidine peptide and it was purified as a p70/p70 homodimer. The two enzymes differed in their specific activity, kinetic properties, and in vitro activation by viral and non-viral proteases. The recombinant His-RT exhibited a lower specific activity than the recombinant RT. The latter yielded enzyme activities as high as an Escherichia coli-expressed RT. Removal of the hexahistidine tag from the recombinant His-RT by digestion with enterokinase resulted in a complete loss of enzyme activity. Thus, the hexahistidine tag might be an intrinsic part of the active recombinant His-RT.
Pekrun
K
K
Department for Virology and Immunology, German Primate Centre, Göttingen, Germany.
Petry
H
H
Jentsch
K D
KD
Moosmayer
D
D
Hunsmann
G
G
Lüke
W
W
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D015153:Blotting, Western; D002478:Cells, Cultured; D002918:Chymotrypsin / Q000378:metabolism; D017931:DNA Primers; D015870:Gene Expression; D005822:Genetic Vectors; D016333:HIV Protease / Q000378:metabolism; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D007313:Insecta; D007700:Kinetics; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D017931:DNA Primers; D011994:Recombinant Proteins; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D002918:Chymotrypsin; D014357:Trypsin; D016333:HIV Protease