Thiol-directed immobilization of recombinant IgG-binding receptors.
A genetic engineering approach is described for directed immobilization of IgG-binding receptors to a thiol-containing matrix using a single cysteine residue. The cysteine residue is introduced into the C-terminal part of receptors based on staphylococcal protein A. Receptors containing one, two or five IgG-binding domains were produced in Escherichia coli and subsequently immobilized on thiopropyl-Sepharose. A high amount, 5 mumol/ml gel (45 mg/ml), of recombinant receptor could be rapidly immobilized to the solid support and both the gel and the immobilized receptor could be regenerated by reduction and oxidation reactions. The gel was used for affinity purification of human IgG and analysis of IgG-binding capacity at different amounts of immobilized recombinant protein show the same maximal IgG-binding capacity (20-25 mg/ml) for all three immobilized receptors. However, at low substitution grade of receptors, the immobilized receptor molecules were shown to bind one (Z-Cys) and two (ZZ-Cys) IgG molecules, respectively. These results demonstrate that the immobilized protein molecules are in a functionally active form and that a two-domain receptor can bind two molecules of IgG without steric hindrance. Interestingly, the five-domain receptor (ZV-Cys), with a structure similar to native protein A, can only bind approximately two IgG molecules, despite the five-domain structure of the molecule.
Ljungquist
C
C
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
Jansson
B
B
Moks
T
T
Uhlén
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000943:Antigens, Differentiation / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D003545:Cysteine; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005813:Genes, Synthetic; D007074:Immunoglobulin G / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011961:Receptors, Fc / Q000235:genetics / Q000378:metabolism*; D017452:Receptors, IgG; D011994:Recombinant Proteins / Q000378:metabolism; D012685:Sepharose; D013205:Staphylococcal Protein A
D000943:Antigens, Differentiation; D007074:Immunoglobulin G; D011961:Receptors, Fc; D017452:Receptors, IgG; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; D012685:Sepharose; D003545:Cysteine