Rapid, high-throughput purification of HIV-1 integrase using microtiter plate technology.
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 microg 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/microg 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.
Wang
Tan
T
Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, NM 88003, USA.
John
Sinu
S
Archuleta
Sergio
S
Jonsson
Colleen B
CB
D018929:Cell Culture Techniques / Q000379:methods; D002847:Chromatography, Agarose; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics; D019427:HIV Integrase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009841:Oligonucleotides / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D003470:Culture Media; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D019427:HIV Integrase