Purification and characterization of human immunodeficiency virus type 1 reverse transcriptase

Stuart F.J. Le Grice, Craig Eugene Cameron, Stephen Benkovic

Research output: Contribution to journalArticle

114 Citations (Scopus)

Abstract

This chapter discusses the purification and characterization of human immunodeficiency virus type 1 reverse transcriptase. Metal chelate chromatography is now finding widespread use in the purification of human immunodeficiency virus (HIV) reverse transcriptase (RT) directly from the high-speed supernatant of bacterial homogenates. Metal chelate affinity chromatography offers a rapid and highly reproducible means of preparing (1) the individual p66 and p51 HIV RT subunits, (2) heterodimer p66/p51, and (3) reconstituted, selectively modified heterodimer directly from bacterial homogenates. The application of a highly selective affinity matrix as the primary purification step has the advantage that bacterial proteases are eliminated at an early stage, avoiding proteolysis of the reconstituted protein. The ease with which HIV RT can be purified by metal chelate affinity chromatography has prompted to develop methodologies for protein mini preparation, where four small columns can be run simultaneously and RT eluted in a batch wise fashion. This procedure has been proven successful in cases where individual domains of the enzyme have been analyzed by insertional mutagenesis.

Original languageEnglish (US)
Pages (from-to)130-144
Number of pages15
JournalMethods in enzymology
Volume262
Issue numberC
DOIs
StatePublished - Jan 1 1995

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HIV Reverse Transcriptase
Purification
Affinity chromatography
HIV-1
Metals
Affinity Chromatography
Proteolysis
Mutagenesis
Insertional Mutagenesis
RNA-Directed DNA Polymerase
Chromatography
Proteins
Peptide Hydrolases
Enzymes
Human immunodeficiency virus 1 reverse transcriptase

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

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Purification and characterization of human immunodeficiency virus type 1 reverse transcriptase. / Le Grice, Stuart F.J.; Cameron, Craig Eugene; Benkovic, Stephen.

In: Methods in enzymology, Vol. 262, No. C, 01.01.1995, p. 130-144.

Research output: Contribution to journalArticle

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