Immobilization of mouse single-chain antibodies for affinity chromatography using the cellulose-binding protein.
A laboratory method for obtaining immunoaffinity medium for chromatographic purification of recombinant human interferon alpha2b (IFN-alpha2b) is described. The method is based on oriented and non-covalent immobilization of recombinant antibody fragments on cellulose. The single-chain fragment variable (ScFv) against human IFN-alpha2b was genetically fused to cellulose-binding domain (CBD) from Clostridium thermocellum cellulosome and expressed in Escherichia coli. After the isolation of the target protein in functionally active form from bacteria cells its bioaffinity immobilization on several forms of cellulose powders has been carried out. The crystalline microgranular cellulose with immobilized ScFv-CBD-fusion protein was used as affinity medium to perform the purification of recombinant human IFN-alpha2b directly from clarified extract of E. coli cells.
Gilchuk
P V
PV
Taras Shevchenko Kyiv National University, Ukraine. Gilchuk@ukr.net
Volkov
G L
GL
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D002482:Cellulose / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D003001:Cloning, Molecular; D003013:Clostridium / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D048168:Escherichia coli K12 / Q000235:genetics; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology; D002479:Inclusion Bodies; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D051379:Mice; D008969:Molecular Sequence Data; D019151:Peptide Library; D010957:Plasmids; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D011994:Recombinant Proteins
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D007128:Immunoglobulin Fragments; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; D002482:Cellulose