Open AccessHuman immunodeficiency virus reverse transcriptase expressed in transformed yeast cells
Author(s) -
SALLAFRANQUEANDREOLA MarieLine,
ROBERT Dominique,
BARR Philip J.,
FOURNIER Michel,
LITVAK Simon,
SARIHCOTTIN Leila,
TARRAGOLITVAK Laura
Publication year - 1989
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1989.tb15028.x
Subject(s) - reverse transcriptase , biology , microbiology and biotechnology , dna polymerase , dna , polymerase , enzyme , yeast , virology , rna , biochemistry , gene
Human immunodeficiency virus (HIV) reverse transcriptase has been purified from yeast transformed by an autoreplicating plasmid containing the retroviral DNA polymerase gene. The previously described purification procedure for the yeast‐expressed reverse transcriptase [Barr, P. J., Power, M. D., Chun Ting Lee‐Ng, Gibson, H. & Luciw, P. (1987) Bio/Technology 5 , 486–489] has been substantially modified, leading to an increased yield and a higher degree of purity. Several biochemical properties of the enzyme are described (template specificity, effect of DNA synthesis inhibitors); interestingly, HIV reverse transcriptase is highly resistant to N ‐ethylmaleimide. A complex between the human retroviral enzyme and the bovine tRNA Lys was shown, using a direct approach, by glycerol gradient centrifugation, as well as by the protective and specific effect of the tRNA Lys against enzyme inactivation by thermal denaturation and trypsin digestion. A competitive type of inhibition of HIV reverse transcriptase by tRNA Lys , but not by tRNA Val , is observed when viral RNA or activated DNA are used as templates.