Immobilized metal affinity chromatography of monoclonal immunoglobulin M against mutant amidase from Pseudomonas aeruginosa |
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Authors: | Sónia Martins Amin Karmali Jorge Andrade Maria Luísa Serralheiro |
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Institution: | 1. Centro de Investiga??o de Engenharia Quimica e Biotecnologia, Instituto Superior de Engenharia de Lisboa Rua Conselheiro Emidio Navarro, 1, 1959-007, Lisboa, Portugal 2. Departmento de Quimica e Bioquimica, Faculdade de Ciências da Universidade de Lisboa, Campo Grade, C8, 1749-016, Lisboa, Portugal
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Abstract: | The chromatographic behavior of monoclonal antibodies (MAbs) of immunoglobulin (Ig) M class against mutant (T103I) amidase
from Pseudomonas aeruginosa was investigated on immobilized metal chelates. The effect of ligand concentration, the length of spacer arm, and the nature
of metal ion were investigated in immobilized metal affinity chromatography (IMAC). The MAbs against mutant amidase adsorbed
to Cu(II), Ni(II), Zn(II), Co(II), and Ca(II)-iminodiacetic acid (IDA) agarose columns. The increase in ligand concentration
(epichlorohydrin: 30–60 and 1,4-butanediol-diglycidyl ether: 16–36) resulted in higher adsorption to IgM into immobilized
metal chelates. The length of spacer arm was found to affect protein adsorption, as longer spacer arm (i.e., 1,4-butanediol-diglycidyl
ether) increased protein adsorption of immobilized metal chelates. The adsorption of IgM onto immobilized metal chelates was
pH dependent because an increase in the binding of IgM was observed as the pH varied from 6.0 to 8.0. The adsorption of IgM
to immobilized metal chelates was the result of coordination of histidine residues to metal chelates that are available in
the third constant domain of heavy chain (CH3) of immunoglobulins, as the presence of imidazole (5 mM) in the equilibration buffer abolished the adsorption of IgM to the column. The combination of tailor-made stationary phases
for IMAC and a correct design of the adsorption parameters permitted to devise a one-step purification procedure for IgM.
Culture supernatants containing IgM against mutant amidase (T103I) were purified either by IMAC on EPI-60-IDA-Co (II) column
or by gel filtration chromatography on Sephacryl S-300HR. The specific content of IgM and final recovery of antibody activity
exhibited similar values for both purification schemes. The purified preparations of IgM obtained by both schemes were apparently
homogeneous on native polyacrylamide gel electrophoresis with a M
r
of 851,000 Da. The results presented in this work strongly suggest that one-step purification of IgM by IMAC is a cost-effective
and process-compatible alternative to other types of chromatography. |
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Keywords: | Monoclonal antibodies IgM class altered (T103I) amidase Pseudomonas aeruginosa immobilized metal affinity chromatography Co2+ ions epichlorohydrin |
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