Matrix-assisted refolding of single-chain Fv- cellulose binding domain fusion proteins.
We describe a method for the isolation of recombinant single-chain antibodies in a biologically active form. The single-chain antibodies are fused to a cellulose binding domain as a single-chain protein that accumulates as insoluble inclusion bodies upon expression in Escherichia coli. The inclusion bodies are then solubilized and denatured by an appropriate chaotropic solvent, then reversibly immobilized onto a cellulose matrix via specific interaction of the matrix with the cellulose binding domain (CBD) moiety. The efficient immobilization that minimizes the contact between folding protein molecules, thus preventing their aggregation, is facilitated by the robustness of the Clostridium thermocellum CBD we use. This CBD is unique in retaining its specific cellulose binding capability when solubilized in up to 6 M urea, while the proteins fused to it are fully denatured. Refolding of the fusion proteins is induced by reducing with time the concentration of the denaturing solvent while in contact with the cellulose matrix. The refolded single-chain antibodies in their native state are then recovered by releasing them from the cellulose matrix in high yield of 60% or better, which is threefold or higher than the yield obtained by using published refolding protocols to recover the same scFvs. The described method should have general applicability for the production of many protein-CBD fusions in which the fusion partner is insoluble upon expression.
Berdichevsky
Y
Y
Department of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat Aviv, 69978, Israel.
Lamed
R
R
Frenkel
D
D
Gophna
U
U
Bayer
E A
EA
Yaron
S
S
Shoham
Y
Y
Benhar
I
I
D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000276:immunology; D000906:Antibodies / Q000737:chemistry; D001665:Binding Sites; D002482:Cellulose / Q000378:metabolism*; D003013:Clostridium / Q000737:chemistry; D005109:Extracellular Matrix; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000302:isolation & purification* / Q000378:metabolism; D002479:Inclusion Bodies; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015202:Protein Engineering; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D012995:Solubility; D001616:beta-Galactosidase / Q000276:immunology
D016229:Amyloid beta-Peptides; D000906:Antibodies; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D002482:Cellulose; D001616:beta-Galactosidase