Rapid, high-throughput purification of HIV-1 integrase using microtiter plate technology

Tan Wang, Sinu John, Sergio Archuleta, Colleen Jonsson

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

The human immunodeficiency virus type-1 (HIV-1) integrase (IN) catalyzes the insertion of the retroviral genome into the chromosome of an infected host cell. HIV-1 IN was expressed as a N-terminal hexa-histidine fusion in Escherichia coli. A high-throughput purification strategy was developed using denaturing methods for the initial protein extraction, followed by a one-step nickel-chelating chromatography purification and step-wise refolding. IN was routinely greater than 90% pure with yields exceeding 14 μg of purified IN per ml of E. coli culture. In vitro 3′ processing and strand transfer assays showed the enzyme preparations to be highly active. The specific activity of the purified IN was 2.65 pmol/h/μg IN, which is very similar to the activity of IN routinely produced by large-scale column chromatographic methods. This high-throughput platform should be of general utility to those interested in defining the structure-function relationship of proteins and enzymes.

Original languageEnglish (US)
Pages (from-to)232-237
Number of pages6
JournalProtein Expression and Purification
Volume33
Issue number2
DOIs
StatePublished - Jan 1 2004
Externally publishedYes

Fingerprint

Integrases
HIV-1
Technology
Escherichia coli
Enzyme Assays
Nickel
Histidine
Chromatography
Proteins
Chromosomes
Genome
Enzymes

All Science Journal Classification (ASJC) codes

  • Biotechnology

Cite this

Rapid, high-throughput purification of HIV-1 integrase using microtiter plate technology. / Wang, Tan; John, Sinu; Archuleta, Sergio; Jonsson, Colleen.

In: Protein Expression and Purification, Vol. 33, No. 2, 01.01.2004, p. 232-237.

Research output: Contribution to journalArticle

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