       Document 0650
 DOCN  M9650650
 TI    Expression and characterization of the reverse transcriptase enzyme from
       type 1 human immunodeficiency virus using different baculoviral vector
       systems.
 DT    9605
 AU    Pekrun K; Petry H; Jentsch KD; Moosmayer D; Hunsmann G; Luke W;
       Department for Virology and Immunology, German Primate Centre,;
       Gottingen, Germany.
 SO    Eur J Biochem. 1995 Dec 15;234(3):811-8. Unique Identifier : AIDSLINE
       MED/96163464
 AB    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.
 DE    Animal  Baculoviridae/*GENETICS  Blotting, Western  Cells, Cultured
       Chymotrypsin/METABOLISM  DNA Primers  Gene Expression  *Genetic Vectors
       Human  HIV Protease/METABOLISM  HIV-1/*ENZYMOLOGY  Insects  Kinetics
       Protein Folding  Protein Processing, Post-Translational  Recombinant
       Proteins/CHEMISTRY/ISOLATION & PURIF/METABOLISM  RNA-Directed DNA
       Polymerase/CHEMISTRY/*GENETICS/ISOLATION & PURIF/  *METABOLISM
       Trypsin/METABOLISM  JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

