A double-modification strategy for enhancing charge density of mono-sized beads for facilitated refolding of like-charged protein

• A double modification strategy was developed to enhance charge density of beads.
• The double modification enhanced BSA adsorption density of the beads.
• Double-modified beads enhanced like-charged protein refolding more significantly.
• Refolding yield was independent of the ligand structure.
• Beads of higher charge density showed lower salt sensitivity in protein refolding.

We have previously found that addition of like-charge media in a refolding system can greatly enhance protein refolding yield, and the media of higher charge density (q) are favorable for facilitating protein refolding. Herein, a double-modification strategy was developed to increase the charge density of mono-sized poly(glycidyl methacrylate) (PGMA) microspheres (0.8 μm). The PGMA beads were firstly modified with poly(ethylenimine) of molecular weight 60,000 (PEIL) or 1200 (PEIS) and then further modified with 2-diethylaminoethylchloride (DEAE) or PEIS. The charge density and bovine serum albumin (BSA) adsorption density (QBSA) of the beads were significantly increased by the double modification. The QBSA of the double-modified beads with the highest q (824 μmol/g) reached 36.3 mg/g, over 35% higher than that of the single-modified beads (26.7 mg/g) with the highest q (300 μmol/g) that a single modification could achieve. The double-modified beads with different ligand structures and charge densities were used for facilitating the refolding of like-charged lysozyme. Lysozyme refolding yield with the single-modified beads of the highest q (300 μmol/g) was 60% at a critical bead concentration (cc) of 100 mg/mL. By contrast, the refolding yields with the double-modified beads of q > 500 μmol/g were 70%, and the refolding yields with the double-modified beads of q ≥ 650 μmol/g could even reach 70% at a cc of 40 mg/mL. This indicates that the double-modified beads of high q values could enhance like-charged protein refolding more significantly than the single-modified beads at a low bead utilization. The facilitating effect of the like-charged beads was independent of the ligand structure. The beads of higher q values showed lower salt sensitivity in protein refolding, and were expected beneficial for use in refolding buffers of higher ionic strengths. The sequential modification strategy for enhancing charge density would help develop more efficient media for protein adsorption and protein refolding applications.