A two plasmid co-expression system in Escherichia coli for the production of virion-like reverse transcriptase of the human immunodeficiency virus type 1.
Many bacterial expression systems have been developed to study the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). This enzyme exists in the virions as a heterodimer of a 66 kDa (p66) subunit and a 51 kDa (p51) subunit, originating through proteolytic maturation of the p66 subunit. Most expression systems rely on the processing of p66 by bacterial proteases, this results in a p51 subunit with a non-authentic carboxy-terminus. In contrast, the expression system described produces an RT with an authentic carboxy-terminus. This was achieved by the co-expression of the two subunits of HIV-1 RT, which were each cloned on a different, compatible plasmid in Escherichia coli, and by the use of protease inhibitors during cell lysis. This approach enabled us not only to obtain virion-like RT, as verified by mass spectrometry, but also to monitor the effect of mutations in one or both subunits on the activity of RT and on its sensitivity towards RT inhibitors. The co-expression system described represents a useful method to produce HIV-1 RT, both authentic and mutated, in quantities that allow large-scale studies on the functional organisation of the RT-subunits and the sensitivity of the enzyme to RT inhibitors.
Jonckheere
H
H
Rega Institute for Medical Research, Katholieke Universiteit Leuven, Belgium.
De Vreese
K
K
Debyser
Z
Z
Vandekerckhove
J
J
Balzarini
J
J
Desmyter
J
J
De Clercq
E
E
Anné
J
J
D001569:Benzodiazepines / Q000494:pharmacology; D003848:Deoxyguanine Nucleotides / Q000494:pharmacology; D054306:Dideoxynucleotides; D004926:Escherichia coli / Q000378:metabolism*; D015870:Gene Expression; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics*; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D013058:Mass Spectrometry; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D013141:Spiro Compounds / Q000494:pharmacology; D013936:Thymidine / Q000031:analogs & derivatives / Q000494:pharmacology; D014771:Virion
D003848:Deoxyguanine Nucleotides; D054306:Dideoxynucleotides; D007093:Imidazoles; C086147:R 86183; D011993:Recombinant Fusion Proteins; D018894:Reverse Transcriptase Inhibitors; D013141:Spiro Compounds; C062687:R 82150; D001569:Benzodiazepines; C082493:1-(2',5'-bis-O-(tert-butyldimethylsilylribofuranosyl)-3-N-methylthymine)-3'-spiro-5''-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide); C031709:2',3'-dideoxyguanosine 5'-triphosphate; D054303:HIV Reverse Transcriptase; D013936:Thymidine