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

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 26 mg for randomly conjugated adsorbent. Remarkably, the high antibody-binding capacity for site-specifically conjugated adsorbent outperformed the existing commercial protein A Sepharose (approximately 30 mg/g). The orientation of a protein is crucial for its activity after immobilization, and these results demonstrate that the site-specifically conjugated protein molecule is in a functionally active form to interact with the antibody with weak steric hindrance. The proposed approach may be an attractive strategy to synthesize affinity adsorbents with high-binding capacity.

SUBMITTER: Zhang X

PROVIDER: S-EPMC6044777 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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

Zhang Xufeng X Duan Ya Y Zeng Xi X

ACS omega 20170428 4

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 ...[more]

PMID: 30023643

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Project description:The lipase from Pseudomonas fluorescens (PFL) has been immobilized on octyl-agarose beads under 16 different conditions (varying pH, ionic strength, buffer, adding some additives) at two different loadings, 1 and 60 mg of enzyme/g of support with the objective of check if this can alter the biocatalyst features. The activity of the biocatalysts versus p-nitrophenyl butyrate and triacetin and their thermal stability were studied. The different immobilization conditions produced biocatalysts with very different features. Considering the extreme cases, using 1 mg/g preparations, PFL stability changed more than fourfolds, while their activities versus pNPB or triacetin varied a 50-60%. Curiously, PFL specific activity versus triacetin was higher using highly enzyme loaded biocatalysts than using lowly loaded biocatalysts (even by a twofold factor). Moreover, stability of the highly loaded preparations was higher than that of the lowly loaded preparations, in many instances even when using 5°C higher temperatures (e.g., immobilized in the presence of calcium, the highly loaded biocatalysts maintained after 24 h at 75°c a 85% of the initial activity, while the lowly loaded preparation maintained only 27% at 70°C). Using the highly loaded preparations, activity of the different biocatalysts versus pNPB varied almost 1.7-folds and versus triacetin 1.9-folds. In this instance, the changes in stability caused by the immobilization conditions were much more significant, some preparations were almost fully inactivated under conditions where the most stable one maintained more than 80% of the initial activity. Results suggested that immobilization conditions greatly affected the properties of the immobilized PFL, partially by individual molecule different conformation (observed using lowly loaded preparations) but much more relevantly using highly loaded preparations, very likely by altering some enzyme-enzyme intermolecular interactions. There is not an optimal biocatalyst considering all parameters. That way, preparation of biocatalysts using this support may be a powerful tool to tune enzyme features, if carefully controlled.

Project description:Site-selective conjugation generally requires both (i) molecular engineering of the protein of interest to introduce a conjugation site at a defined location and (ii) a site-specific conjugation technology. Three N-terminal interferon α2-a (IFN) variants with truncated histidine tags were prepared and conjugation was examined using a bis-alkylation reagent, PEG(10kDa)-mono-sulfone 3. A histidine tag comprised of two histidines separated by a glycine (His2-tag) underwent PEGylation. Two more IFN variants were then prepared with the His2-tag engineered at different locations in IFN. Another IFN variant was prepared with the His-tag introduced in an α-helix, and required three contiguous histidines to ensure that two histidine residues in the correct conformation would be available for conjugation. Since histidine is a natural amino acid, routine methods of site-directed mutagenesis were used to generate the IFN variants from E. coli in soluble form at titres comparable to native IFN. PEGylation conversions ranged from 28-39%. A single step purification process gave essentially the pure PEG-IFN variant (>97% by RP-HPLC) in high recovery with isolated yields ranging from 21-33%. The level of retained bioactivity was strongly dependent on the site of PEG conjugation. The highest biological activity of 74% was retained for the PEG10-106(HGHG)-IFN variant which is unprecedented for a PEGylated IFN. The His2-tag at 106(HGHG)-IFN is engineered at the flexible loop most distant from IFN interaction with its dimeric receptor. The biological activity for the PEG10-5(HGH)-IFN variant was determined to be 17% which is comparable to other PEGylated IFN conjugates achieved at or near the N-terminus that have been previously described. The lowest retained activity (10%) was reported for PEG10-120(HHH)-IFN which was prepared as a negative control targeting a IFN site thought to be involved in receptor binding. The presence of two histidines as a His2-tag to generate a site-selective target for bis-alkylating PEGylation is a feasible approach for achieving site-selective PEGylation. The use of a His2-tag to strategically engineer a conjugation site in a protein location can result in maximising the retention of the biological activity following protein modification.

Dataset Information

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

Publications

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

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