Immunoaffinity chromatography in biorecovery: an application of recombinant DNA technology to generic adsorption processes.
The constant region of human kappa light chain (Ck) was linked to Escherichia coli beta-galactosidase, using standard molecular cloning techniques. The binding of Ck-beta-galactosidase fusions to a number of different murine monoclonal antibodies, specific for Ck, was improved by the insertion of spacers between Ck and beta-galactosidase: a cleavable linker was then introduced. Over-expressed Ck-beta-galactosidase fusion protein was purified using monoclonal antibodies immobilised on Sepharose 4B. Elution conditions were found that maintained beta-galactosidase activity so purified enzyme could be released on breaking the cleavable linker. A number of practical problems associated with maintaining stable fusion proteins and immunoaffinity column performance were identified.
Downham
M
M
School of Chemical Engineering, University of Birmingham, Edgbaston, UK.
Busby
S
S
Jefferis
R
R
Lyddiatt
A
A
D000327:Adsorption; D000911:Antibodies, Monoclonal / Q000276:immunology; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D006801:Humans; D006898:Hydroxylamines; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000302:isolation & purification; D007145:Immunoglobulin kappa-Chains / Q000235:genetics / Q000302:isolation & purification; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification*; D012685:Sepharose / Q000031:analogs & derivatives / Q000737:chemistry; D001616:beta-Galactosidase / Q000737:chemistry
D000911:Antibodies, Monoclonal; D006898:Hydroxylamines; D007143:Immunoglobulin Heavy Chains; D007145:Immunoglobulin kappa-Chains; D011994:Recombinant Proteins; C035771:Sepharose CL 4B; D012685:Sepharose; D001616:beta-Galactosidase