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. 2017;52(2):276-286.
doi: 10.1080/01496395.2016.1206934. Epub 2016 Aug 17.

Affinity membrane adsorbers for binding arginine-rich proteins

Heather C S Chenette  1 , James M Welsh  1 , Scott M Husson  1
Affiliations

Affinity membrane adsorbers for binding arginine-rich proteins

Heather C S Chenette et al. Sep Sci Technol. 2017.

Abstract

Delivering protein chemotherapeutics into cancer cells is a challenge. Fusing the protein to an arginine-rich cell-penetrating peptide offers a possible solution. The goal of this work was to develop an affinity membrane for purification of Arg-rich fusion proteins via capture chromatography. Membranes were prepared by grafting polymers bearing diethyl-4-aminobenzyl phosphonate (D4ABP) ligands from macroporous membrane supports. Incorporation of D4ABP was studied by infrared spectroscopy and energy dispersive spectroscopy. Protein binding capacities of 3 mg lysozyme/mL were measured. While further studies are required to evaluate binding kinetics and Arg-selectivity, achieving higher protein binding capacity is needed before investment in such studies.

Keywords: ATRP; Arg-tag; fusion protein; membrane chromatography; surface modification.

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Figures

Figure 1
Figure 1
ATR-FTIR spectra of unmodified RC60 regenerated cellulose membrane and membrane after ATRP-grafted PGMA, which contains a peak at 1730 cm−1 assigned to C=O.
Figure 2
Figure 2
ATR-FTIR spectra of conditions of A, B, C, D, E, F. Scan of condition D contains a peak near 1500 cm−1.
Figure 3
Figure 3
ATR-FTIR spectra of conditions G, H, I, and J
Figure 4
Figure 4
ATR-FTIR spectra of membranes modified under specified conditions. A presents conditions H2 and D2 for 2h and 43h, an insert (B) is given for better view. C presents condition J2 for times 4h to 123h. C shows entire scan, an insert (D) is given for better view.
Figure 5
Figure 5
Dry layer thickness values. The model used literature values for the RI of all materials except for ATRP PGMA and D4ABP PGMA that were obtained from the model. (See Figure 6.) Error bars show the standard deviation of three measurements within one Si wafer chip.
Figure 6
Figure 6
Refractive indices obtained with combined layer model. Error bars represent the standard deviation in the three measurements.
Figure 7
Figure 7
SEM images of PGMA-modified membrane surfaces after J2 reaction at various times. A = 0 h, B = 16 h, C = 123 h. Images are common scale, with scale bar shown below image A.
Scheme 1
Scheme 1
Reaction of D4ABP with ATRP-grafted PGMA
See this image and copyright information in PMC

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