Figure 1.
Illustration of membrane fabrication and recycling.
Molecular structure of PP2b, schematic depiction of supramolecular fibers, 3D network, and membrane. Hydrophobic groups of PP2b are located in the fibers’ core (red), whereas their hydrophilic PEG shell (blue) provides a biocompatible interface. Recycling of the membrane is achieved by disaggregation or physical removal of the supramolecular layer from the support, followed by purification, and subsequent reassembly in aqueous solution.
Figure 2.
Stable flux of aqueous solutions over time.
Flux of water, NaCl(aq), and MOPS buffer solution through a freshly prepared supramolecular membrane.
Figure 3.
Cryo-SEM images of the supramolecular PP2b membrane on the cellulose acetate (CA) support, which had been permeated by MOPS buffer solution.
Table 1.
Size-related values of the proteins: calculated molecular weight (MW), hydrodynamic diameter (Dh), and molecular dimensions estimated from X-ray structures.
Figure 4.
Filtration of protein mixtures.
(A) UV/Vis spectra of the protein mixture before and after filtration over the pristine CA membrane. (B) Representative UV/Vis spectra of the protein mixture before filtration through the supramolecular membrane, the filtrate (collected in 5×1.5 ml fractions, F1–F5), and filtered buffer solution as a reference (F0) (C) Total protein concentration in the filtrate fractions F1–F5 as compared to the feed solution, determined from absorbance at 280 nm. Error bars correspond to the standard deviation of 5 independent filtration experiments.
Figure 5.
Separation of protein mixtures.
(A) Typical SDS-PAGE used for densitometric quantification of individual protein concentrations. MWM = molecular weight marker (170, 130, 95, 72, 55, 43, 34, 26, 17, 11 kDa). (B) Concentrations of individual proteins in fractions F1–F5 (normalized with respect to the non-filtered solution), and recycled proteins. (C) Protein retention against molecular weight (black data points) and sigmoid fit (red curve). Protein structures of KE70 (PDB;3Q2D), Aldolase (PDB;1DZU) and CS hexamer (PDB;1NXG) are shown. (D) Dependence of protein retention on the hydrodynamic diameter (black data points) and sigmoid fit (red curve). All error bars represent the standard deviation of 5 independent filtration experiments.
Figure 6.
Gel filtration chromatogram of a mixture of BSA oligomers and monomers before filtration (black trace), and its filtrate (red trace). Filtration quantitatively removes BSA oligomers (≥ 400 kDa, retention time: 7 min) from the mixture. Smaller BSA aggregates (retention time: 11–12 min) are removed as well. The filtrate contains pure monomeric proteins (∼67 kDa, retention time: 13 min).
Figure 7.
Kinetics of the KE70 activity.
Change in absorbance at 380 nm before filtration, after filtration, and of neat buffer solution, following addition of 5-Nitrobezisoxazole (at t = 0 min).
Figure 8.
Heterogeneous biocatalysis in the supramolecular membrane using immobilized β-Gal.
(A) Hydrolysis of ONPG into Galactose and ONP. (B) Yield of ONP as a function of time during several hours of continuous flux of substrate.
Figure 9.
Conversion of Oxaloacetate and Acetyl-CoA into Citrate and HS-CoA over CS immobilized in a PP2b supramolecular membrane.
HS-CoA reacts with DTNB in the assay solution to release the indicator of the reaction, TNB (λmax = 412 nm). The color change from clear reactant feed solution to yellow filtrate indicates biocatalytic activity of the immobilized enzymes.