Expression, purification and applications of staphylococcal protein A fused to cellulose-binding domain.
Because staphylococcal Protein A (ProtA) binds specifically to IgG, it has been used for many immunological manipulations, most notably antibody purification and diagnostics. Immobilization is required for most of these applications. Here we describe a genetic-engineering approach to immobilizing ProtA on cellulose, by fusing it to cellulose-binding domain (CBD) derived from the cellulose-binding Protein A of Clostridium cellulovorans. The bifunctional fusion protein was expressed in Escherichia coli, recovered on a cellulose column and purified by elution at alkaline pH. ProtA-CBD was used to purify IgG from rabbit serum and its ability to bind IgG from different sources was determined. The bifunctional chimaeric protein can bind up to 23.4 mg/ml human IgG at a ratio of 1 mol of ProtA-CBD/2 mol of human IgG, and can purify up to 11.6 mg/ml rabbit IgG from a serum. The ability to bind functionally active CBD-affinity reagents to cellulosic microtitre plates was demonstrated. Our results indicate that a combination of CBD-affinity reagents and cellulosic microtitre plates is an attractive diagnostics matrix for the following reasons: (i) cellulose exhibits very low non-specific binding; and (ii) CBD-fusion proteins bind directly to cellulose at high density. A unique signal-amplification method was developed based on the ability of ProtA-CBD to link stained cellulose particles to primary antibody in a Western blot.
Shpigel
E
E
The Kennedy Leigh Centre for Horticulture Research and The Otto Warburg Center for Agricultural Biotechnology, The Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot, Israel.
Goldlust
A
A
Eshel
A
A
Ber
I K
IK
Efroni
G
G
Singer
Y
Y
Levy
I
I
Dekel
M
M
Shoseyov
O
O
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007074:Immunoglobulin G / Q000302:isolation & purification* / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D019809:Streptavidin / Q000276:immunology; D014462:Ultrafiltration / Q000295:instrumentation / Q000379:methods
D001426:Bacterial Proteins; D002352:Carrier Proteins; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; C074074:cellulose binding protein A, Clostridium cellulovorans; D002482:Cellulose; D019809:Streptavidin