One-step capillary isoelectric focusing for the separation of the recombinant human immunodeficiency virus envelope glycoprotein glycoforms.
One-step capillary isoelectric focusing was investigated as a rapid method to resolve the glycoforms of the heterogeneous recombinant human immunodeficiency virus (HIV) envelope glycoprotein (rgp 160sMN/LAI). The separation was performed in a poly(vinyl alcohol) (PVA) coated capillary using a mixture of ampholyte of narrow and wide pH range. A combination of saccaharose and 3-(cyclohexylamino)-1-propanesulfonic acid was shown to be the most efficient additive to avoid protein precipitation which occurs at a pH close to its pI. Although the calibration curve [isoelectric point (pI) vs. migration times] showed a non-linear relationship, an adequate linearity could be yielded for short pI ranges permitting to exhibit the acidic character of the different glycoforms of the rgp 160s MN/LAI (pI from 4.00 to 4.95). Reproducibility evaluated by comparing the performance of a polyacrylamide and a PVA coated capillary showed that low RSD values were obtained for intra-day (0.5 to 1.9%) and inter-day (1.6 to 7.6%) measurements using the PVA capillary. Moreover, the long term stability of the PVA capillary was demonstrated by measuring the variation of migration times of the protein markers for a long period of use. Finally, this method was able to differentiate the glycoform pattern of two close glycoproteins such as the rgp 160 of two sub-populations of the virus HIV-1.
Tran
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Laboratoire de Chimie Analytique, Faculté de Pharmacie, Châtenay-Malabry, France.
Taverna
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Chevalier
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Ferrier
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D019075:Electrophoresis, Capillary / Q000379:methods*; D019302:HIV Envelope Protein gp160 / Q000737:chemistry / Q000302:isolation & purification*; D015497:HIV-1 / Q000737:chemistry*; D007525:Isoelectric Focusing / Q000379:methods*; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000302:isolation & purification; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet
D019302:HIV Envelope Protein gp160; D020033:Protein Isoforms; D011994:Recombinant Proteins