Improved separation of integral membrane proteins by continuous elution electrophoresis with simultaneous detergent exchange: application to the purification of the fusion protein of the human immunodeficiency virus type 1.
We describe a protocol for preparative-scale purification of the fusion protein of the human immunodeficiency virus type 1 (HIV-1), gp41, from cells overexpressing the viral envelope proteins and from HIV-1 isolates. In the first step, the proteins were extracted from the membrane in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. The extract was then subjected to separation by continuous elution electrophoresis using a nonionic or zwitterionic detergent in the mobile elution buffer, which results in the simultaneous exchange of SDS with that detergent. The separated proteins were obtained in an SDS-free buffer containing either Brij, 3-[(3-chloramidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or Triton X-100 and could then be subjected to subsequent purification steps like isoelectric focusing in the second dimension or immunoaffinity chromatography. The dilute protein fraction was concentrated and applied on a 10 mL immunoaffinity column packed with anti-gp41 monoclonal antibody immobilized on protein-G sepharose. The protein was eluted from the column at pH 2.7 and obtained in pure form in amounts of 30-50 micrograms that constituted a yield of 1%. The pure gp41 could not be sustained in solution in the absence of detergent and was not susceptible to proteolytic digestion by trypsin. The identification of the protein and the degree of purity was confirmed indirectly using surface enhanced laser desorption ionization-time of flight-mass spectrometry (SELDI-TOF-MS). The possible application of this approach for the isolation of integral membrane proteins with the propensity to undergo spontaneous folding and aggregation is being discussed.
Laboratory of Experimental and Computational Biology, Center for Cancer Research, NCI-Frederick, Bldg. 469, Rm. 213, Miller Drive, Frederick, MD 21702, USA.
Mathias
M
Viard
Robert
R
Blumenthal
Yossef
Y
Raviv
D002846:Chromatography, Affinity / Q000379:methods; D003902:Detergents; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D015700:HIV Envelope Protein gp41 / Q000032:analysis / Q000302:isolation & purification*; D015497:HIV-1 / Q000737:chemistry; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods; D008565:Membrane Proteins / Q000032:analysis / Q000302:isolation & purification*; D017830:Octoxynol
D003902:Detergents; D015700:HIV Envelope Protein gp41; D008565:Membrane Proteins; D017830:Octoxynol