Improved Performance of Recombinant Protein A Immobilized on Agarose Beads by Site-Specific Conjugation

ACS Omega
Xufeng ZhangXi Zeng

Abstract

Protein A affinity adsorbent with high antibody-binding capacity plays a prominent part in the purification of biopharmaceuticals to decrease the manufacturing costs. We describe a site-specific covalent conjugation strategy for protein A to immobilize on agarose beads. Recombinant protein A, which has one cysteine introduced at the C terminus through genetic engineering technology, was immobilized site-specifically on maleimide-functionalized agarose beads by the thiol-maleimide reaction. As a comparison, the recombinant protein A was randomly immobilized on the aldehyde-functionalized agarose beads via free amino groups on the protein surface. The site-specific conjugation of recombinant protein A on the agarose beads was validated through the assay of free SH groups on the adsorbents using the Ellman's reagent. Adsorbents containing various amounts of protein A were used to adsorb antibody from human plasma. Analysis of immunoturbidimetry showed that the adsorbed fractions contained the 90.1% IgG, 4.2% IgA, and 5.7% IgM. The maximal antibodies-binding capacities with static adsorption and dynamic adsorption were approximately 64 and 50 mg, respectively, per swollen gram for site-specifically conjugated adsorbent and 31 and 2...Continue Reading

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Citations

Dec 8, 2020·Analytical Sciences : the International Journal of the Japan Society for Analytical Chemistry·Shigenori OtaShuichi Yamamoto

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Methods Mentioned

BETA
electrophoresis

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