Immobilization of Carboxymethylated Polyethylenimine–Metal-Ion Complexes in Porous Membranes to Selectively Capture His-Tagged Protein

Abstract

Membrane adsorbers rapidly capture tagged proteins because flow through membrane pores efficiently conveys proteins to binding sites. Effective adsorbers, however, require membrane pores coated with thin films that bind multilayers of proteins. This work employs adsorption of polyelectrolytes that chelate metal ions to create functionalized membranes that selectively capture polyhistidine-tagged (His-tagged) proteins with binding capacities equal to those of high-binding commercial beads. Adsorption of functional polyelectrolytes is simpler than previous membrane-modification strategies such as growth of polymer brushes or derivatization of adsorbed layers with chelating moieties. Sequential adsorption of protonated poly(allylamine) (PAH) and carboxymethylated branched polyethylenimine (CMPEI) leads to membranes that bind Ni(2+) and capture ∼60 mg of His-tagged ubiquitin per mL of membrane. Moreover, these membranes enable isolation of His-tagged protein from cell lysates in <15 min. The backbone amine groups in CMPEI likely increase swelling in water to double protein binding compared to films composed of PAH and the chelating polymer poly[(N,N-dicarboxymethyl)allylamine] (PDCMAA), which has a hydrocarbon backbone. Metal leaching from PAH/CMPEI- and PAH/PDCMAA-modified membranes is similar to that from GE Hitrap FF columns. Eluates with 0.5 M imidazole contain <10 ppm of Ni(2+).

Carboxymethylated polyethylenimine modified magnetic nanoparticles specifically for purification of His‐tagged protein

“…From the FTIR spectral of CMPEI (Figure B), the characteristic peaks at 1750 and 1650 cm −1 arise from the C=O stretching and asymmetric stretching in the NH + ‐CH 2 COOH and NH + ‐CH 2 COO group, respectively. Meanwhile, for the FTIR spectrum of CMPEI@SiO 2 @Fe 3 O 4 , the absorption peak at 1750 cm −1 disappeared and the peak around 1650 cm −1 enhanced, which demonstrated that most of carboxylate groups on CMPEI are deprotonated . Besides, the peaks at 598 and 1060 cm −1 are respectively corresponding to the stretching vibration of Fe‐O and Si‐O‐Si of CMPEI@SiO 2 @Fe 3 O 4 .…”

“…As shown in Supporting Information Figure S2, the binding capacity of CMPEI 3 ‐Ni 2+ @SiO 2 @Fe 3 O 4 made with high molecular weight CS is lower than that of CMPEI‐Ni 2+ @SiO 2 @Fe 3 O 4 , indicating the usage of high molecular weight CS could not display the advantage of LBL assembly technique. This phenomena could be attributed to the existence of numerous salt bridges between protonated amine groups of CS and some COO‐ groups of CMPEI, which consumed a certain amount of docking sites and hinder Hexa His‐tagged peptides to contact these sites . Therefore, the low molecular weight CS was used in the present work unless otherwise specified.…”

“…As shown in Figure S5, the adsorption amount of the sixth usage could remain over 75% of the initial binding amount, which suggested the magnetic nanoparticles could be used repeatedly. The reason of performance decline was related with the surface metal‐ion leaching of magnetic nanoparticles during the operation process . Besides, a small loss of magnetic nanoparticles exists during the operations of binding, elution and washing, resulting in the decrease of adsorption capacity.…”

“…Subsequently, the resulting SiO 2 ‐NH 2 @Fe 3 O 4 nanoparticles were collected with a magnet and washed with ethanol, water, and then dried under vacuum at room temperature. CMPEI was synthesized as described in the published reports . Then, 30 mg of SiO 2 ‐NH 2 @Fe 3 O 4 was ultrasonically dispersed in a solution containing 3 mg/mL CMPEI and 0.1 M NaCl at pH 5.…”

“…Moreover, CMPEI contains abundant IDA groups, which has been utilized to adsorb metal ions such as Cu 2+ , Co 2+ , Ni 2+ , and Zn 2+ . When charging with metal ions, CMPEI immobilized in porous membranes was also used to capture His‐tagged protein . In this study, a facile synthesis strategy to prepare a novel kind of magnetic IMAC material named as CMPEI‐Ni 2+ @SiO 2 @Fe 3 O 4 was developed by using LBL technique with CMPEI as an affinity ligand.…”