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An alternative for purification of low soluble recombinant hepatitis C virus core protein: Preparative two‐dimensional electrophoresis
Author(s) -
Yvon Stéphane,
Rolland Dominique,
Charrier JeanPhilippe,
Jolivet Michel
Publication year - 1998
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150190814
Subject(s) - isoelectric focusing , chromatography , electrophoresis , recombinant dna , chemistry , gel electrophoresis , protein purification , polyacrylamide gel electrophoresis , isoelectric point , two dimensional gel electrophoresis , immunogenicity , sodium dodecyl sulfate , escherichia coli , biochemistry , biology , enzyme , antibody , proteomics , immunology , gene
For isolation of low soluble recombinant full‐length (amino acids 1–191) core protein of hepatitis C virus (HCV) overexpressed in Escherichia coli , the advantage of combining two electrophoretic techniques, in comparison with chromatographic separation, is demonstrated. The protein extract was first solubilized in agents compatible with electrophoretic separation. Using preparative liquid phase isoelectric focusing (IEF) the protein of interest was first concentrated within a defined acidic pH range. These fractions were then submitted to preparative sodium dodecyl sulfate ‐ polyacrylamide gel electrophoresis (SDS‐PAGE) to isolate the 22 kDa protein. The second‐dimensional step allowed the isolation of 2 mg of the purified recombinant HCV core protein (rHCV‐C191) from 1.5 g bacterial pellet. This quantity is sufficient to characterize the protein and to perform immunogenicity studies. This procedure of two‐dimensional preparative electrophoresis is applicable to a wide range of biological samples and represents an alternative for purification of insoluble proteins.