Extraction of HIV-1 in aqueous two-phase systems to obtain a high yield of gp120.
The conventionally applied centrifugation protocols for the concentration and purification of human immunodeficiency virus type 1 (HIV-1) result in a low recovery of the external glycoprotein, gp120. This is consistent with what has been found with other retroviruses. In the search for a method allowing the copurification of the gp120 with the virion we have applied two-phase extraction based on water-soluble polymers. Several polymer systems were tested for their capacity to enrich HIV-1 from HTLV-IIIB-infected H9 cell culture medium. With a dextran-polyethylene glycol system the gp120 and the gag protein p24, used as marker of the virion, were recovered to about 60 and 70%, respectively, in 1% of the initial volume. The two proteins were both about 30-fold purified and reverse transcriptase activity and infectious titer were retained to a high degree. The calculated molar ratio of gp120 to p24 was twofold higher in the phase-extracted fraction than in material pelleted by ultracentrifugation. It is concluded that extraction in aqueous two-phase systems is a method well suited for the concentration and initial purification of HIV-1. The technique is adaptable to almost any scale and may replace ultracentrifugation. Qualitatively, its main advantage over the latter method is the enhanced recovery of the gp120 in the virion fraction.
Hammar
L
L
Department of Veterinary Virology, Biomedical Center, Uppsala, Sweden.
Gilljam
G
G
D002460:Cell Line; D003470:Culture Media; D015699:HIV Envelope Protein gp120 / Q000302:isolation & purification*; D015497:HIV-1 / Q000302:isolation & purification*; D006801:Humans; D008722:Methods; D011108:Polymers; D012995:Solubility
D003470:Culture Media; D015699:HIV Envelope Protein gp120; D011108:Polymers