Polyacid-functionalized porous membranes, related methods, and related polyacid polymers
11654402 · 2023-05-23
Assignee
Inventors
- Merlin L. Bruening (East Lansing, MI, US)
- Salinda Wijeratne (East Lansing, MI, US)
- Wenjing Ning (East Lansing, MI, US)
- Jinlan Dong (East Lansing, MI, US)
- Weijing Liu (East Lansing, MI, US)
Cpc classification
B01D71/64
PERFORMING OPERATIONS; TRANSPORTING
B01D67/0088
PERFORMING OPERATIONS; TRANSPORTING
B01D71/38
PERFORMING OPERATIONS; TRANSPORTING
B01D71/82
PERFORMING OPERATIONS; TRANSPORTING
B01D69/141
PERFORMING OPERATIONS; TRANSPORTING
C08L77/02
CHEMISTRY; METALLURGY
B01D69/02
PERFORMING OPERATIONS; TRANSPORTING
B01D71/40
PERFORMING OPERATIONS; TRANSPORTING
International classification
B01D67/00
PERFORMING OPERATIONS; TRANSPORTING
B01D69/02
PERFORMING OPERATIONS; TRANSPORTING
B01D71/38
PERFORMING OPERATIONS; TRANSPORTING
B01D71/40
PERFORMING OPERATIONS; TRANSPORTING
B01D71/64
PERFORMING OPERATIONS; TRANSPORTING
B01D71/82
PERFORMING OPERATIONS; TRANSPORTING
Abstract
The disclosure relates to processes, related polyacid polymers, and related articles for functionalizing a porous membrane by contacting the membrane with a polyacid polymer at low pH to stably adsorb a polyacid layer on the membrane pore surface, in particular polyacid polymers including repeating units with a pendent metal-binding ligand or star polyacid polymers. The resulting functionalized membrane is characterized by a high density of free acid groups, resulting in a higher specific capacity for its intended application. The process allows functionalization of porous membranes in a very simple, one-step process, for example without a need to derivatize an adsorbed polyacid layer to impart metal-binding ligand functionality thereto. Such functional membranes may find multiple uses, including rapid, selective binding of proteins for their purification or immobilization.
Claims
1. A polyacid-coated porous membrane comprising: (a) a porous membrane substrate comprising a plurality of membrane pores; and (b) a polyacid layer adsorbed on surfaces of the membrane pores, the polyacid layer comprising a polyacid polymer comprising repeating units comprising a pendent metal-binding ligand comprising one or more free acid groups selected from the group consisting of carboxylic acid groups, carboxylate groups, and combinations thereof; wherein: the polyacid layer is stably adsorbed on the surfaces of the membrane pores and is substantially free of covalent attachments to the surfaces of the membrane pores; and the repeating units are selected from the group consisting of: (i) repeating units comprising a nitrogen atom (N) present in a backbone portion of the repeating units; (ii) repeating units comprising (A) an amide group (—C(═O)NH— or —C(═O)NR—) linking the pendent metal binding ligand and the polyacid polymer backbone, and (B) a pendent alkyl or heteroalkyl group on the polyacid polymer backbone at the same location as the amide group; and (iii) repeating units comprising an oxygen atom (O) present in a backbone portion of the repeating units.
2. The polyacid-coated porous membrane of claim 1, wherein the polyacid layer is stably adsorbed on the surfaces of the membrane pores due to one or more of hydrophobic interactions, hydrogen bonding interactions, and coordination interactions.
3. The polyacid-coated porous membrane of claim 1, further comprising: (c) metallic ions complexed with the metal-binding ligands.
4. The polyacid-coated porous membrane of claim 3, wherein the metallic ions comprise one or more of Ni.sup.2+, Cu.sup.2+, Co.sup.2+, Fe.sup.3+, and Ga.sup.3+.
5. The polyacid-coated porous membrane of claim 1, wherein: (i) the repeating units comprise the nitrogen atom and have the general formula —[—CH.sub.2—CH.sub.2—NR—]—; and (ii) R comprises the pendent metal-binding ligand.
6. The polyacid-coated porous membrane of claim 1, wherein: (i) the repeating units comprise the amide linking group and have the general formula —[—CH.sub.2—CR.sub.1(C(═O)NR.sub.2R)—]—; (ii) R comprises the metal-binding ligand; (iii) R.sub.1 is a C.sub.1-C.sub.4 alkyl or heteroalkyl group; and (iv) R.sub.2 is H or a C.sub.1-C.sub.4 alkyl or heteroalkyl group.
7. The polyacid-coated porous membrane of claim 1, wherein: (i) the repeating units comprise the oxygen atom and have the general formula —[—O—CH.sub.2—CH(CH.sub.2NR)—]—; and (ii) R comprises the metal-binding ligand.
8. The polyacid-coated porous membrane of claim 1, wherein the plurality of membrane pores has an average pore size ranging from 0.02 μm to 50 μm.
9. The polyacid-coated porous membrane of claim 1, wherein the porous membrane substrate comprises a synthetic polymeric membrane material selected from the group consisting of cellulose acetates, nitrocelluloses, cellulose esters, polysulfones, polyether sulfones, polyacrylonitriles, polyamides, polyimides, polyethylenes, polypropylenes, polytetrafluoroethylenes, polyvinylidene fluorides, polyvinylchlorides, hydroxylated derivatives of the foregoing, and combinations thereof.
10. The polyacid-coated porous membrane of claim 1, wherein the polyacid layer is adsorbed directly on the porous membrane substrate.
11. The polyacid-coated porous membrane of claim 1, wherein the polyacid layer is immobilized on the porous membrane substrate via one or more adhesion layers, wherein at least one of the adhesion layers is adsorbed directly on the porous membrane substrate.
12. The polyacid-coated porous membrane of claim 1, wherein the polyacid-coated porous membrane has a monolayer of the polyacid polymer adsorbed directly on the porous membrane substrate and comprising the free acid groups.
13. The polyacid-coated porous membrane of claim 1, wherein the polyacid-coated porous membrane substrate comprises a plurality of polyacid layers, wherein (i) a first polyacid layer is adsorbed directly on the porous membrane substrate and (ii) one or more further polyacid layers are adhered to adjacent polyacid layers via one or more intervening polycation layers.
14. The polyacid-coated porous membrane of claim 1, wherein the metal-binding ligands comprise one or more of nitrilotriacetic acid groups, iminodiacetic acid groups, and salts thereof.
15. The polyacid-coated porous membrane of claim 1, wherein: (i) the repeating units comprise the nitrogen atom and have the general formula —[—(CR.sub.aR.sub.b).sub.n—NR—]—; (ii) n is 2, 3, or 4; (iii) R.sub.a is H or a C.sub.1-C.sub.4 alkyl or heteroalkyl group; (iv) R.sub.b is H or a C.sub.1-C.sub.4 alkyl or heteroalkyl group; and (v) R comprises the metal-binding ligand and a C.sub.2-C.sub.16 alkyl or heteroalkyl group.
16. The polyacid-coated porous membrane of claim 1, wherein: (i) the repeating units comprise the oxygen atom and have the general formula —[—O—(CR.sub.aR.sub.b).sub.n—CH(R.sub.cNR)—]—; (ii) n is 1, 2, or 3; (iii) R.sub.a is H or a C.sub.1-C.sub.4 alkyl or heteroalkyl group; (iv) R.sub.b is H or a C.sub.1-C.sub.4 alkyl or heteroalkyl group; (iv) R.sub.c is a C.sub.1-C.sub.4 alkyl or heteroalkyl linking group; and (v) R comprises the metal-binding ligand and a C.sub.2-C.sub.16 alkyl or heteroalkyl group.
17. A method for binding an affinity-tagged target protein, the method comprising: (a) providing the polyacid-coated porous membrane according to claim 3; (b) providing a feed fluid sample comprising a target protein comprising an affinity tag; and (c) passing the feed fluid sample through the polyacid-coated porous membrane, thereby (i) binding at least some of the target protein via the affinity tag with the immobilized protein affinity tag-binding ligands and (ii) providing a permeate fluid with at least some of the target protein removed.
18. The method of claim 17, wherein (i) the affinity tag is a polyhistidine tag, and (ii) the metallic ions comprise one or more of Ni.sup.2+ and Co.sup.2+.
19. The method of claim 17, further comprising: (d) eluting the bound target protein from the polyacid-coated porous membrane, thereby forming a purified permeate comprising the target protein.
20. The method of claim 17, wherein (i) the feed fluid sample further comprises non-target proteins, and (ii) the purified permeate is substantially free from the non-target proteins.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) For a more complete understanding of the disclosure, reference should be made to the following detailed description and accompanying drawings wherein:
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DETAILED DESCRIPTION
(23) Typical membrane modification includes polymerization from the surface. This is a relatively complex process, and often includes initiator attachment to the membrane. The current approach involves adsorption of a polyacid to the membrane. Hydrophobic interactions strongly attach the polymer to the surface. Although others have modified membranes through polymer adsorption, a feature of the disclosed process is adsorption at low pH to maintain a low fraction of ionized groups and promote the formation of highly swollen films after deprotonation of the acid groups. These highly swollen films rapidly bind large amounts of protein and can be further functionalized. The method is much more convenient than previous approaches to membrane modification. Protein binding capacities are higher than for commercial membranes. In some embodiments, the adsorbed polyacid polymers incorporate metal-binding ligands as deposited onto the membrane surface, thus eliminating the need for further post-adsorption functionalization processes. In some embodiments, the adsorbed polyacid polymers incorporate nitrogen and/or oxygen heteroatoms into their otherwise carbon-based polymer backbone, which heteroatoms can improve film swelling and, correspondingly, membrane capture capacity and efficiency for a target analyte.
(24) The process involves simple passage of a polyacid solution through a membrane at low pH. Additional layers may be deposited by sequentially adsorbing polycations along with the polyanion at low pH. Subsequent binding at neutral pH leads to a high density of ion-exchange sites for protein binding. Derivatization of the acid groups with ligands such as Ni.sup.2+ complexes allows selective binding of tagged proteins such as those containing polyhistidine.
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(29) Similar to
EXAMPLES
(30) The following examples illustrate the disclosed processes and compositions, but are not intended to limit the scope of any claims thereto.
(31) The polyacid polymer CMPEI of Example 1 was synthesized and adsorbed on a membrane to promote swelling by protonation of amine groups at neutral pH, for example the amino nitrogen atoms along the polymer backbone. Control over pH permits high swelling and creates membranes that capture large amounts of protein. Membranes containing protonated poly(allylamine)/CMPE-Cu.sup.2+ complexes bind 60 mg of protein per mL of membrane. If Ni.sup.2+ is present, such membranes capture His-tagged protein from cell extracts.
(32) The polyacid copolymer of Example 4 includes acrylic acid and optionally acrylamide comonomers along with monomer repeat units having pendent metal-binding NTA ligands. The acrylic acid repeat units should promote swelling when deprotonated, and the units containing NTA will bind Ni.sup.2+ to capture His-tagged protein. PAH/poly(NTA-co-AA-ACM) films can be formed with high thicknesses.
(33) Example 5 describes a new method for making a polyacid polymer or copolymer with monomer repeat units having pendent metal-binding NTA ligands and optionally acrylic acid monomer units. The synthetic method greatly simplifies preparation, and metal leaching appears to be lower with this system. The acrylic acid comonomers can help control swelling. Films (4 to 5 bilayers) of this polymer on solid substrates swell well (−400-600%) and bind large amounts of Cu.sup.2+ (2.0 mmol/cm.sup.3). Membranes modified with these polymers also bind large amounts of metal ions. Preliminary studies of absorption of His-tagged protein Pantoea agglomerans (phenylalanine aminomutase (PaPAM), MW 55-60k) shows films containing two bilayers of poly(allylamine)/poly(NTA-Am)—Ni.sup.2+ complexes bind 4 multilayers of protein. Membranes with Cu.sup.2+ complexes capture 45 mg/mL of Con A.
(34) Example 6 describes star polyacid polymers. Layer-by-layer deposition of these star polyacid polymers alternating with a corresponding star polycation polymer gives unique porous structures that may be especially valuable for sorption. These films show high swelling (˜300-500%) and membranes containing star PAA/PDMAEMA bind 90-105 mg of Lysozyme per mL of membrane. The porous structure may facilitate protein transport into the film for subsequent binding.
(35) Example 7 describes polyethylene oxide-based polyacid polymers. Polymers with polyethylene glycol backbones can be synthesized as shown in the example. Ideally, a more hydrophilic, oxygen-rich backbone can introduce more swelling to the polyelectrolyte films. Also, these polyethylene glycol polymer backbones are widely used in protein purification systems to reduce nonspecific binding. Since both polymer-backbones (cationic and anionic) contain polyethylene glycol backbone, more swelling could be possible with these polyelectrolytes.
Example 1: CMPEI-Functionalized Membranes
(36) Membrane adsorbers rapidly capture tagged proteins because convective mass transport through membrane pores efficiently conveys proteins to binding sites. Fabrication of effective adsorbers, however, involves modification of membrane pores with thin films that swell in water and bind multilayers of proteins. This example demonstrates membrane modification via adsorption of polyelectrolytes that chelate metal ions, and these modified membranes selectively capture polyhistidine-tagged proteins with capacities higher than those of commercial beads. Direct adsorption of functional polyelectrolytes is simpler than prior 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 polyethyleneimine (CMPEI) leads to membranes that bind Ni.sup.2+ and capture 60±6 mg of his-tagged ubiquitin per mL of membrane. Moreover, the membrane enables isolation of his-tagged protein from cell lysates in as little as 20 min. Although it contains both cationic and anionic groups, CMPEI acts as a polyanion during polyelectrolyte adsorption. The cationic groups in CMPEI potentially increase swelling in water compared to films composed of PAH and the chelating polymer poly[(N,N-dicarboxymethyl) allylamine] (PDCMAA), which has a hydrocarbon backbone. Consistent with higher swelling, PAH/CMPE-Cu.sup.2+ films capture almost twice as much protein as PAH/PDCMAA-Cu.sup.2+ films. Metal leaching from PAH/CMPEI- and PAH/PDCMAA-modified membranes is similar to that from GE HITRAP FF columns, presumably because of related chelating groups in the three materials. Eluates with 0.5 M imidazole contain <10 ppm Ni.sup.2+.
(37) In most studies of overexpressed proteins, purification employs engineered affinity tags. Hexahistidine is the most common affinity tag because it is relatively small and enables convenient capture by binding to beads containing Ni.sup.2+ or C.sup.2+ complexes. Nevertheless, bead-based separations suffer from slow diffusion of large macromolecule into nanopores, which necessitates long separation times that may harm sensitive proteins. Separations are especially time consuming when capturing proteins from large volumes of dilute solutions. Porous membranes modified with affinity ligands are an attractive alternative purification platform because convection through the membrane pores and short radial diffusion distances provide rapid protein transport to binding sites. Moreover, membrane pressure drops are low because of small thicknesses. Nevertheless, membranes have a lower specific surface area than nanoporous beads, which often leads to a low binding capacity.
(38) To increase protein-binding capacities of membranes, several groups modified membrane pores with thin polymer films. Both surface-initiated growth of polymer brushes and layer-by-layer polyelectrolyte adsorption can provide highly swollen films that capture multiple layers of proteins. Compared to the synthesis of polymer brushes, which is a relatively cumbersome process that frequently requires initiator immobilization and subsequent polymerization under anaerobic conditions, layer-by-layer deposition is quite simple. Layer-by-layer adsorption of (polyacrylic acid) PAA/(polyethyleneimine) (PEI) films and subsequent derivatization with aminobutyl nitrilotriacetate (NTA) to immobilize NTA-Ni.sup.2+ complexes on a membrane surface can be used to capture his-tagged proteins. However, derivatization represents more than 95% of the cost of chemicals and materials for creating protein-binding membranes, and most of the aminobutyl NTA does not couple to the membrane. In addition to NTA these membranes contain other-COOH groups that bind Ni.sup.2+ only weakly, which leads to significant metal-ion leaching and loss of the complexed metallic ions from the membrane.
(39) This illustrates the direct adsorption of relatively inexpensive polyelectrolytes with chelating groups (Scheme 1.1) that can effectively create functionalized membranes 104 that bind metal ions and capture his-tagged protein P (
(40) Materials:
(41) PCDMAA was synthesized as described in Wijeratne (2013) and in Example 2. CMPEI was synthesized as illustrated in Scheme 1.1 and described in Example 3. Aqueous solutions containing 0.02 M PAH, 0.01 M CMPEI or 0.01 M PDCMAA were prepared in deionized water (18.2 MΩcm, Milli-Q) or 0.5 M aqueous NaCl, and solution pH values were adjusted by drop-wise addition of 0.1 M NaOH or HCl. Polymer concentrations are given with respect to the repeating unit. Au-coated silicon wafers (200 nm of sputtered Au on 20 nm of Cr on Si (100) wafers) were cleaned in a UV/O.sub.3 chamber for 15 min prior to use. Other materials include hydroxylated nylon (LOPRODYNE LP, Pall, 1.2 μm pore size, 110 μm thick), conconavalinA (Con A) from Canavalia ensiformis (Jack bean), coomassie protein assay reagent (Thermo Scientific), histidine6-tagged ubiquitin (His-U, human recombinant, Bostonbiochem), polyethyleneimine (PEI, branched, Mw=25 000), poly(allylamine hydrochloride) (PAH, molecular weight 120,000-200,000 Da, Alfa Aesar), and poly(acrylic acid) (PAA, Mw=90 000, 25% aqueous solution, Polysciences). Cupric sulfate, nickel sulfate, sodium phosphate, sodium phosphate dibasic, ethylenediaminetetraacetic acid disodium salt (EDTA), sodium chloroacetate (98%), 3-mercaptopropionic acid (MPA, 99%) and imidazole (>99%) were received from Aldrich and used without further purification. Buffers include: Binding buffer 1: 20 mM phosphate buffer, pH 6; Binding buffer 2: 20 mM phosphate buffer, pH 7.4; Washing buffer 1: 20 mM phosphate buffer, 150 mM NaCl, 1% TWEEN 20, pH 7.4; Washing buffer 2: 20 mM phosphate buffer, 45 mM imidazole, 150 mM NaCl, pH 7.4; Elution buffer: 20 mM phosphate buffer, 500 mM NaCl, 500 mM imidazole, pH 7.4; Stripping buffer: 20 mM phosphate buffer, 500 mM NaCl, 50 mM EDTA, pH 7.4; All experiments were repeated two or three times, and uncertainty values are standard deviation of three experiments with independent membranes.
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(43) Adsorption of Polyelectrolyte Multilayers (PEMs):
(44) Au-coated Si substrates (24 mm×11 mm) were immersed in 5 mM MPA in ethanol for 16 h, rinsed with ethanol, and dried with N.sub.2 to form a monolayer of MPA for adsorption of PAH. These substrates were immersed in 0.02 M PAH (adjusted to the desired pH) for 15 min and subsequently rinsed with 10 mL of deionized water and blown dry with N.sub.2. Substrates were then immersed in a 0.01 M CMPEI or PDCMAA solution (adjusted to the desired pH value) for 15 min followed by the same rinsing and drying procedures. In some cases, the polyelectrolyte solution also contained 0.5 M NaCl. The process was repeated to form multilayer films.
(45) In some cases, nylon membranes were immersed in 0.1 M sodium chloroacetate in 3 M NaOH for 16 h and subsequently washed with water and dried with N.sub.2. The resulting carboxymethylated membrane disks were cleaned for 10 min with UV/O.sub.3 and placed in a homemade TEFLON (PTFE) holder (similar to an AMICON cell) that exposed 3.1 cm.sup.2 of external membrane surface area. Subsequently, a 5 mL solution containing 0.02 M PAH and 0.5 M NaCl was circulated through the membrane for 15 min at a flow rate of 1 mL/min using a peristaltic pump. A CMPEI or PDCMAA layer was deposited similarly using 0.01 M CMPEI or 0.01 M PDCMAA solutions containing 0.5 M NaCl. After deposition of each polyelectrolyte layer, 20 mL of water was passed through the membrane at the same flow rate. Nylon membranes without carboxymethylation were modified with PEMs similarly, starting with the UV/O.sub.3 cleaning.
(46) Characterization of Polyelectrolyte Film on Gold Wafers:
(47) Spectroscopic ellipsometry (model M-44; J. A. Woollam) was used to determine the thicknesses of PEMs on gold-coated wafers, assuming a film refractive index of 1.5. Film thickness in aqueous solutions was measured in a home-built cell. In that case, the software determines the refractive index of swollen films. Reflectance FTIR spectra were obtained with a Thermo Nicolet 6700 FTIR spectrometer using a Pike grazing angle (80°) apparatus. A UV/ozone-cleaned Au-coated wafer served as a background.
(48) Metal and Protein Binding in (PAH/CMPEI).sub.n- and (PAH/PDCMAA).sub.n-Modified Wafers and Membranes:
(49) Bare carboxymethylated nylon and membranes containing (PAH/CMPEI).sub.n and (PAH/PDCMAA).sub.n were loaded with Cu.sup.2+ or Ni.sup.2+ by circulating 0.1 M CuSO.sub.4 or NiSO.sub.4 (pH around 3.8) through the membrane for 30 min, followed by rinsing with 20 mL water. Metal was eluted from the membranes by stripping buffer or 2% HNO.sub.3 and subsequently analyzed by atomic absorption spectroscopy.
(50) For protein capture on wafers coated with PEMs, the modified substrates were immersed in solutions containing 0.3 mg/mL of Con A or His U in binding buffer 1 for Con A and binding buffer 2 for His U for 1 h at room temperature. Subsequently, using a Pasteur pipette these substrates were rinsed with 10 mL of washing buffer 1 and 10 mL of water for 1 min each and dried with N.sub.2. The amount of protein binding was determined by reflectance FTIR spectroscopy and expressed as the equivalent thickness of spin-coated protein that would give the same absorbance. The equivalent thickness d is calculated from the difference in absorbance (ΔA) at 1680 cm.sup.−1 (amide band I of protein) before and after binding, using the equation d(nm)=ΔA/0.0017. Some of these thicknesses were confirmed using ellipsometry. Assuming a protein density of 1 g/cm.sup.3, each nm of equivalent thickness corresponds to approximately 1 mg/m.sup.2 of surface coverage.
(51) Breakthrough curves for capture in membranes were obtaining by passing protein solution (0.3 mg/mL) in binding buffer 1 or binding buffer 2 through the membranes. For Con A and lysozyme binding, these studies employed the homemade TEFLON holder that exposed 3.1 cm.sup.2 of external membrane surface area. Because of the high cost of his-tagged protein, TEFLON holder was used that exposed a surface area of 0.78 cm.sup.2 (1.0-cm exposed diameter) to test the protein binding capacity for His-U. Bradford assays (using calibration with the protein of interest) were employed to quantify the concentration of proteins in the membrane effluent or eluate.
(52) Protein Separation from a Cell Extract:
(53) His-tagged small ubiquitin modifier (His-SUMO) was over-expressed in E. coli cells. The cells were lysed with sonication in binding buffer 2 and centrifuged. Supernatant (2 mL) was pumped thorough the (PAH/CMPEI)-modified membrane at room temperature at a flow rate of 1 mL/min. Subsequently the membrane was rinsed with 5 mL of binding buffer 2 and 5 mL of washing buffer 2, and the bound protein was eluted with 2 mL of elution buffer. The purity of the eluted protein was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
(54) Metal Leaching and Film Stability:
(55) To examine film stability under purification conditions, (PAH/CMPEI).sub.2-modified gold wafers were soaked in binding buffer 2 for 20 hours. Film thickness values and FTIR spectra were obtained before after immersion in the buffer for different times. TOC analysis was used to quantify the amount of polyelectrolyte leaching from membranes in binding buffer 2. Polyelectrolyte solutions with concentration of 0 ppm, 10 ppm, 20 ppm, 30 ppm, 40 ppm and 50 ppm were used for TOC calibration. The effluent and washing solutions were diluted with water before analysis.
(56) To test the metal leaching in different buffers, (PAH/PDCMAA)-, (PAH/PDCMAA).sub.2-, (PAH/CMPEI)- and (PAH/CMPEI).sub.2-modified carboxymethylated nylon membranes were loaded with Ni.sup.2+ using the above procedure and washed with 160 bed volumes (5 mL) each of binding buffer 2, wash 1, wash 2, elution buffer, stripping buffer, and 2% HNO.sub.3. As a comparison, a GE Healthcare HITRAP IMAC FF column (1 mL) was washed with 160 bed volumes (160 mL) of the same buffers. All the samples were diluted 1:5 with deionized water and analyzed by atomic absorption Healthcare HITRAP spectrometry. The GE IMAC FF column was loaded with Ni.sup.2+ following the manufacture procedure with little modification: loading with 2 mL of 0.1 M NiSO.sub.4 followed by washing with 20 mL deionized water.
(57) Layer-by-Layer Adsorption of Films Containing CMPEI:
(58) CMPEI contains both weakly basic (amine) and weakly acidic (carboxylic acid) groups and thus can potentially form salt bridges with both cations and anions on a surface. An acid titration of CMPEI suggests nearly complete protonation of amine groups below pH 7, whereas protonation of the carboxylate groups begins below pH 4, which is similar to the titration of PDCMAA. This is reasonably consistent with the pKa values for iminodiacetic acid, which are 9.4, 2.6, and 1.8. The ratio of carboxylic acid groups to amines is around 1:1 in CMPEI but 2:1 in iminodiacetic acid and PDCMAA.
(59) Based on the polymer titration and 1:1 ratio of amine to carboxylic acid groups, CMPEI might serve as a polyanion in films formed at basic pH and as a polycation in films formed at acidic pH. With branched CMPEI, adsorption of (polycation/CMPEI).sub.n coatings also occurs at low pH.
(60) During adsorption, carboxylate groups on CMPEI most likely bind to ammonium groups of PAH with displacement of counterions from these groups. Reflectance IR spectroscopy confirms that most of the carboxylate groups in these films are deprotonated (
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(62) The reflectance IR spectra of (PAH/CMPEI)5 films deposited at different pH values show that most of the carboxylic groups are deprotonated. However, the ratio of the absorbances of the —COO— symmetric stretch (˜1650 cm.sup.−1) and the —COOH peak (1720 cm.sup.−1) decreases as the deposition pH decreases.
(63) CMPEI gives very thin films when serving as a polycation in layer-by-layer adsorption. (CMPEI/PSS).sub.5 films deposited at pH 3 in 0.5 M salt have thicknesses of only 10±2 nm. The positive charges of CMPEI reside mostly in or near the backbone and may be less available for adsorption than —COO— groups on the side chains. Using a cyclic analogue of linear CMPEI, Hoffman and Tieke (2009) also found minimal growth during layer-by-layer deposition with PSS.
(64) Film Swelling:
(65) For thin films that selectively bind proteins in platforms such as porous membranes, film swelling in water is important for extensive protein capture. To examine swelling, in situ ellipsometry was performed with (PAH/CMPEI).sub.5 films (deposited at pH 3 from 0.5 M NaCl) immersed in deionized water or binding buffer 2 (pH 7.4). After a 20-minute immersion, swelling of the film was 160±30% in deionized water and 680±260% in buffer. Consistent with the approximately 62% and 88% water in the immersed coatings, the film refractive indices decrease from 1.50 to 1.39 and 1.35 after swelling in water and buffer, respectively. The refractive index of water at the wavelengths of the spectroscopic ellipsometer is about 1.333. Deprotonation of —COOH groups in pH 7.4 buffer likely enhances the swelling, which should provide the space for binding multilayers of protein in the film. IR spectra confirm the deprotonation after immersing the film in buffer. As a comparison, the swelling of (PAH/PDCMAA).sub.5 films (deposited at pH 3 from 0.5 M NaCl) was 52±16% in deionized water and 220±20% in binding buffer 2. The high swelling of (PAH/CMPEI).sub.5 relative to (PAH/PDCMAA).sub.5 suggests that the charged backbone and branched structure of CMPEI facilitate swelling.
(66) Modification of porous membranes to bind proteins can involve adsorption of only a few polyelectrolyte bilayers to simplify the process and avoid plugging of pores. Moreover the films should contain metal-ion complexes for capture of proteins through metal-ion affinity interactions. Thus swelling of (PAH/CMPEI).sub.2 and (PAH/PDCMAA).sub.2 films containing Cu.sup.2+ complexes also was examined. These studies employed binding buffer 1 (pH 6.0) to match subsequent Con A-binding studies, as Con A solutions are not stable at pH 7.4.
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(68) Protein Binding to (PAH/CMPE).sub.2-Cu.sup.2+ and (PAH/PDCMAA).sub.2-Cu.sup.2+ Films:
(69) In initial studies of protein binding, Con A was captured in (PAH/CMPEI).sub.2—Cu.sup.2+ and (PAH/PDCMAA).sub.2-Cu.sup.2+ films on Au-coated Si wafers modified with MPA. Binding likely occurs when histidine groups on the protein coordinate with immobilized Cu.sup.2+. Using reflectance IR spectroscopy, the amount of protein binding was determined based on the amide absorbance, which is compared to the absorbance in spin-coated films with different thicknesses. (PAH/PDCMAA).sub.2-Cu.sup.2+ films have average thicknesses ranging from 7-25 nm, depending on the deposition pH (see
(70) Adsorption of (PAH/CMPEI).sub.2 at deposition pH values from 3-7 leads to thinner films than adsorption at pH 2 and binding of only 5 nm or less of protein (
(71) Adsorption of (PAH/CMPEI).sub.n and (PAH/PDCMAA).sub.n films within membrane pores is difficult to quantify. To qualitatively assess the amount of adsorbed polymer, the Cu.sup.2+ binding in membranes modified with polyelectrolyte films was examined. As
(72) The PAH/CMPEI CM nylon membrane binds 16 times the amount of Cu.sup.2+ captured in a PAH/CMPEI nylon membrane. SEM images of bare nylon, CM nylon, (PAH/CMPEI)—Cu.sup.2+ CM nylon and (PAH/CMPEI).sub.2—Cu.sup.2+ CM nylon (not shown) indicated that the structures of the nylon membranes exhibit no obvious change after carboxymethylation, so the primary effect of this treatment is the formation of —COOH groups that facilitate adsorption of the initial PAH layer.
(73) Selective capture of his-tagged proteins typically employs immobilized Ni.sup.2 or Co.sup.2+ complexes, not Cu.sup.2+. Histidine binding to Ni.sup.2+ and Co.sup.2+ is weaker than to Cu.sup.2+ and thus requires multiple histidines for protein capture, which affords selective capture of his-tagged species. As
(74) Lysozyme Binding in (PAH/CMPEI)-Modified Membranes as a Function of Flow Rate:
(75) Capture rates are important when purifying sensitive proteins or when processing purification large volume of dilute samples. To assess mass-transport limitations on binding, the capture of lysozyme at two different flow rates was examined. The —COOH groups in (PAH/CMPEI) can serve as cation-exchange sites for rapid adsorption of lysozyme (pl ˜11), so binding kinetics likely will not limit the capture rate.
(76) The small non-zero concentration at the beginning of the breakthrough curve is likely due to a small amount of polymer that leaches from the membrane and interferes with the Bradford assay.
(77) Con A Binding to Membranes Modified with PAH/PDCMAA-Cu.sup.2+ and PAH/CMPEI-Cu.sup.2+ Films:
(78) Due to the high cost of his-tagged proteins, Con A binding to Cu.sup.2+ complexes was first used to evaluate the protein-binding capacity of membranes containing PAH/CMPEI and PAH/PDCMAA films.
(79) Con A binding in (PAH/CMPEI).sub.2—Cu.sup.2+-modified CM nylon also was tested. Based on breakthrough curves, the Con A binding capacity in these membranes is 39±5 mg/mL, or less than in membranes with PAH/CMPEI-Cu.sup.2+ films. The unexpected decrease in binding compared to a film with a single bilayer might reflect decreased swelling with more bilayers or limited access to some small pores after coating the spongy membrane structure (see
(80) Capture of His-Tagged Protein Using Membranes Containing PAH/CMPEI-Ni.sup.2+ Films:
(81) Because it showed the highest binding capacity for Con A, the binding capacity for his-tagged ubiquitin using a CM nylon membrane modified with a PAH-CMPEI film was measured. However, in this case, the Ni.sup.2+ complex was used, which is necessary for selective capture of his-tagged protein. Based on both the breakthrough curve and the amount of protein eluted from the membrane, the binding capacity is 60±6 mg/mL (
(82) To demonstrate that membranes can isolate his-tagged protein directly from cell extracts, his-tagged SUMO protein that was over-expressed in E. coli, was purified.
(83) Film Stability:
(84) The stability of CMPEI-containing films both on wafers and in membranes was evaluated. For (PAH/CMPEI).sub.2 films on gold-coated Si wafers (deposited at pH 2 in 0.5 M NaCl) immersion for 20 h in binding buffer 2 (pH 7.4) led to only a 10% decrease in thickness, most of which occurred in the first 4 h (see
(85) Membranes offer a high surface area for film formation, so even a small amount of polyelectrolyte leaching might affect a Bradford assay. Using TOC analysis, the amount of the polyelectrolyte film lost during passage of binding buffer 2 (pH 7.4) through a membrane immediately after forming a (PAH/CMPEI) film and rinsing with only water was determined. The first 20 mL of washing buffer contained around 4 ppm of polymer (assuming that the leaching was only due to CMPEI and using 1-10 ppm CMPEI solutions as standards). Subsequent buffer washes contained <0.1 ppm of polymer. Additionally, wash solutions were added to the Bradford dye and tested the absorbance at 595 nm as in a typical Bradford assay. The first mL of washing solution gave an absorbance of 0.02, which is equivalent to the absorbance given by 0.03 mg/mL Con A. This absorbance rapidly declines and was only 0.002 after passing 20 mL of washing buffer through the membrane.
(86) In a typical protein-binding test, the membranes are washed with 40 mL of binding buffer prior to loading protein. However, breakthrough curves such as those in FIGS. 11 and 13 show a small and decreasing Bradford assay signal over the first 1-2 mL. This may indicate that protein replaces a small amount of polyelectrolyte, i.e., the initial loading solution might contain 5 ppm of polyelectrolyte after passing through the membrane. This was not observed in binding of His U.
(87) As a further test of membrane stability, 5 cycles of loading and elution of Con A in (PAH/CMPEI)—Cu.sup.2+-modified membrane were performed with Con A. Based on either breakthrough curves or elution, the Con A binding decreased by <20% over five cycles of loading and elution.
(88) Metal Leaching:
(89) Low metal-ion leaching is sometimes important to avoid contaminating protein solutions. Thus, metal leaching was examined from a common commercial Ni.sup.2+ column and several modified membranes. Membranes modified with one and two bilayers of PAH/CMPEI or PAH/PDCMAA (deposited at pH 2 in 0.5 M NaCl) were washed with 5 mL each (160 bed volumes) of binding buffer 2, washing buffer 1, and washing buffer 2, stripping buffer and 2% HNO.sub.3. The GE HITRAP FF Ni column with 1-mL bed volume was washed with 160 bed volumes each of binding buffer 2 and washing buffers 1 and 2 and 15 mL of stripping buffer. All the solutions were analyzed by the atomic absorption spectroscopy.
(90)
(91) Summary:
(92) This example illustrates a facile method, layer-by-layer adsorption of functional polyelectrolytes, to modify membranes with metal-ion complexes that selectively capture his-tagged proteins. PAH/CMPEI adsorption yields a membrane with a his-tagged ubiquitin binding capacity of 60±6 mg/mL, which is higher than related commercial affinity membranes and most commercial beads. Moreover, these (PAH/CMPEI)-modified membranes show less than 10 ppm of Ni.sup.2+ in the elution buffer (0.5 M imidazole). Membranes and wafers modified with PAH/CMPEI show about twice the protein binding of corresponding substrates modified with PAH/PDCMAA, presumably because of more swelling with PAH/CMPEI. The his-tagged protein binding capacity of the (PAH/PEI)-modified membranes is only 2/3 of that of membranes modified through growth polymer brushes of layer-by-layer adsorption of PAA/PEI/PAA followed by derivatization. However, direct adsorption of PAH and CMPEI in membranes is much simpler and less expensive than previous membrane modification methods.
Example 2: Synthesis of PDCMAA
(93) The following example describes the synthesis of polyacid polymers having pendent metal binding ligands according to the disclosure.
(94) Synthesis of poly[(N,N-dicarboxymethyl)allylamine]:
(95) Synthesis of poly[(N,N-dicarboxymethyl)allylamine] (PDCMAA) was carried out according to the procedure of Naka (1995) with slight modifications. Under a nitrogen (N.sub.2) atmosphere, chloroacetic acid (6.69 g, 0.07 mol), NaOH (2.80 g, 0.07 mol) and 25 ml of water were added to a two-neck round-bottomed flask, and the mixture was stirred at 30° C. for 10 min. This solution was added drop-wise with stirring to an aqueous solution (100 mL) containing poly(allyamine hydrochloride) (PAH, Mn˜5.8×10.sup.4 Da, 1.0 g, 0.011 mol) at 50° C. The reaction mixture was kept at 50° C. for 1 h and then held at 90° C. for 2 h with occasional addition of 30% NaOH to maintain the pH at 10.0. The reaction mixture was stored at room temperature for 12 h, and then the pH was adjusted to 1 by adding concentrated HCl. The supernatant was decanted, the remaining precipitate was dissolved by addition of 30% NaOH, and the solution was again adjusted to pH 1.0 with concentrated HCl. This process was repeated 2 times, and the precipitate was filtered and dried in vacuo for 12 h. The procedure is illustrated in Scheme 2.1. The resulting white poly[(N,N-dicarboxymethyl)allylamine] (PDCMAA) solid (70% yield) was characterized by .sup.1H-NMR and FTIR spectroscopy. IR (KBr): 1631, 1735 and 1400 cm.sup.−1; .sup.1H-NMR 0.50-2.00 (br, s, 3H), 2.00-2.75 (br s, 2H), 2.80-3.50 and (br s, 4H).
(96) ##STR00002##
Example 3: Synthesis of CMPEI
(97) The following example describes the synthesis of polyacid polymers having pendent metal binding ligands according to the disclosure, in particular those having backbone nitrogen atoms.
(98) Synthesis of Carboxymethylated Polyethyleneimine (CMPEI):
(99) Synthesis of CMPEI was carried out according to the procedure described above and illustrated in Scheme 1.1. Under a nitrogen (N.sub.2) atmosphere, sodium chloroacetate (20.0 g, 0.25 mol) and 25 ml of water were added to a two-neck round-bottomed flask, and the mixture was stirred at 30° C. for 10 min. This solution was added drop-wise with stirring to an aqueous solution (100 mL) containing poly(ethyleneimine) (PEI, M.sub.n˜6.0×10.sup.4 Da, 5.0 g, 10.6 mmol) at 50° C. The reaction mixture was kept at 50° C. for 1 h and then held at 90° C. for 2 h with occasional addition of 30% NaOH to maintain the pH at 10.0. The reaction mixture was stored at room temperature for 12 h, and then the pH was adjusted to 1 by adding concentrated HCl. The supernatant was decanted, the remaining precipitate was dissolved by addition of 30% NaOH, and the solution was again adjusted to pH 1.0 with concentrated HCl. This process was repeated 3 times, and the precipitate was filtered and dried in vacuo for 12 h. The resulting white carboxymethylated polyethyleneimine (CMPEI) solid (63% yield) was characterized by FTIR spectroscopy IR (KBr): 1655 (COO.sup.−) and 1733 (COOH) cm.sup.−1; and Elemental analysis (%) calculated for C.sub.42H.sub.72N.sub.10O.sub.24: C, 45.82; H, 6.59; N, 12.72. Found: C, 40.26; H, 6.65; N, 11.93. The difference between the experimental and calculated elemental analysis stems from the presence of HCl salts in the actual structure.
Example 4: Synthesis of Poly(NTA)-Based Polyacid Copolymers
(100) The following example describes the synthesis of polyacid polymers having pendent metal binding ligands according to the disclosure, in particular those having an ethylenic polymer backbone and pendent nitrilotriacetic free acid groups as metal binding ligands linked to the backbone via an amide group and copolymerized with other acidic comonomers.
Synthesis of 2,2′-((1-carboxy-4-methacrylamidobutyl)azanediyl)diacetic acid (NTA-MAm)
(101) 2,2′-((1-carboxy-4-methacrylamidobutyl)azanediyl)diacetic acid (NTA-MAm) was synthesized according to the procedure of Ehrbar (2008) with slight modifications. First, Nα′,Nα-bis(carboxymethyl)-L-lysine (2.0 g, 7.2 mmol) was dissolved in a 0.4 M NaOH solution (50 ml) and cold the mixture to 0° C. using a ice bath. Then, ice-cold methacryloyl chloride (8.0 mmol, 0.90 ml) in 50 ml toluene was added to the abovementioned solution. Reaction was carried out at 0° C. to room temperature for 24 h. The reaction was monitored with .sup.1HNMR and additional addition of methacryloyl chloride needed for completion of this reaction. Toluene was decanted and an aqueous layer was concentrated to get the final 2,2′-((1-carboxy-4-methacrylamidobutyl)azanediyl)diacetic acid with 1.8 g (Yield 76%). The procedure is illustrated in Scheme 4.1. The resulting compound was characterized using .sup.1HNMR (D.sub.2O, d ppm): 1.39 (m, 2H, CH.sub.2), 1.47 (m, 2H, CH.sub.2), 1.78 (s, 3H, CH3), 1.82-1.88 (m, 2H, CH.sub.2), 3.14 (t, 2H, CH.sub.2), 4.0 (t, 2H, CH.sub.2), 4.0 (s, 2H, 2×CH.sub.2), 5.29 (s, 1H, CH═CCH3[trans]), 5.52 (d, 1H, CH═CCH3[cis])
(102) ##STR00003##
(103) Synthesis of poly(NTA-MAm-co-AA):
(104) First, 2,2′-((1-carboxy-4-methacrylamidobutyl)azanediyl)diacetic acid (NTA-MAm) (0.15 mmol, 0.42 g) and acrylic acid (AA) (0.85 mmol, 0.62 g) were dissolved in 50 ml of 50 mM Tris/HCl buffer, previously adjusted to pH 8.5. Then, five freeze/thaw cycles were performed. Polymerization was initiated by the addition of 150 μl ammonium peroxodisulphate (10%, w/v) and 24 ml of N,N,N,N-tetramethylethylenediamine. Reaction was carried out for 48 h at room temperature. At the end, the solution pH was adjusted to a value of 1.0 by adding concentrated HCl to eliminate low-molecular-weight compounds such as residual acrylic acid. The supernatant was decanted, the remaining precipitate was dissolved by addition of 30% NaOH, and the solution was again adjusted to pH 1.0 with concentrated HCl. This process was repeated 2 times, and the precipitate was filtered and dried in vacuo for 12 h. The procedure is illustrated in Scheme 4.2. The resulting white poly(2,2′-((1-carboxy-4-methacrylamidobutyl)azanediyl)diacetic acid-co-acrylic acid) solid, 0.85 g (Yield 85%) was characterized using .sup.1HNMR (D.sub.2O, d ppm): 0.89 (br, 3H), 1.19-1.31 (br, 2H, CH2), 1.42 (br, 2H, CH2), 1.84 (br, 4H, 2×CH2), 1.90-1.98 (br, 2H, CH2), 2.10 (br, 1H, CH), 2.93 (br, 2H, CH2), 3.50 (br, 5H, 2×CH2 and CH), 7.87 (br, 1H, CONH).
(105) ##STR00004##
(106) Synthesis of poly(NTA-MAm-co-AA-ACM):
(107) 2,2′-((1-carboxy-4-methacrylamidobutyl)azanediyl)diacetic acid (NTA-MAm) (0.15 mmol, 0.42 g), acrylic acid (0.65 mmol, 0.47 g), and acrylamide (2.0 mmol, 0.14 mmol) were dissolved in 50 ml of 50 mM Tris/HCl buffer, previously adjusted to pH 8.5. Then, five freeze/thaw cycles were performed. Polymerization was initiated by the addition of 150 μl ammonium peroxodisulphate (10%, w/v) and 24 μl N,N,N,N-tetramethylethylenediamine. Reaction was carried out for 48 h at room temperature. At the end, the solution pH was adjusted to a value of 1.0 by adding concentrated HCl to eliminate low-molecular-weight compounds such as residual acrylic acid and acrylamide. The supernatant was decanted, the remaining precipitate was dissolved by addition of 30% NaOH, and the solution was again adjusted to pH 1.0 with concentrated HCl. This process was repeated 2 times, and the precipitate was filtered and dried in vacuo for 12 h. The procedure is illustrated in Scheme 4.3. The resulting white poly(2,2′-((1-carboxy-4-methacrylamidobutyl)azanediyl)diacetic acid-co-acrylic acid, acrylamide) solid, 0.50 g (Yield 50%) was characterized using 1HNMR (D.sub.2O, d ppm): 0.95 (br, 3H), 1.41 (br, 4H, 2×CH2), 1.59 (br, 6H, 3×CH2), 2.1 (br, 2H, 2×CH), 2.95 (br, 2H, CH2), 3.62 (br, 5H, 2×CH2 and CH).
(108) ##STR00005##
Example 5: Synthesis of Poly(NTA)-Based Polyacid Copolymers
(109) The following example describes the synthesis of polyacid polymers having pendent metal binding ligands according to the disclosure, in particular those having an ethylenic polymer backbone and pendent nitrilotriacetic free acid groups as metal binding ligands linked to the backbone via an amide group and optionally copolymerized with other acidic comonomers.
(110) ε-Acryloyl L-Lysine Copper Complex:
(111) L-Lysine hydrochloride (20 g, 109.5 mmol) was dissolved in water (250 ml) at 90° C. Basic cupric carbonate (13.3 g, 60.2 mmol) was added slowly to the solution and stirred for 10 min. After cooling and filtering the insoluble residue, 120 ml of acetone was added. After 55 ml of 2.0 M KOH aqueous solution were added into solution of the copper complex of the L-lysine, acryloyl chloride (1.38 ml, 17.1 mmol) and 7.7 ml of 2.0 M KOH aqueous solution were added every 5 min at 0° C. This procedure was repeated eight times. After stirring for 12 h at room temperature, the precipitates of the acrylamide cupric complex of the L-lysine were filtered and washed successively with water, methanol, and ether. The yield was 18 g (71%). This structure was confirmed by their IR spectra. IR (cm.sup.−1): 3000-3500, nN—H (amide, amine); 2890-2900, nC—H; 1660, C═O (amide I); 1630, N—H (amide II); Elemental analysis (%) calculated for C.sub.18H.sub.30N.sub.4O.sub.6Cu: C, 46.80; H, 6.55; N, 12.13. Found: C, 45.82; H, 6.76; N, 12.03.
(112) ε-Acryloyl L-Lysine (LysAm): The solid of c-Acryloyl L-Lysine Copper Complex (5.0 g, 11 mmol) was dispersed in water (100 ml), and a chloroform solution (100 ml) of 8-hydroxyquinolinol (2.0 g, 13.6 mmol) was added. After stirring the solution for 12 h in a Erlenmeyer flask, a green precipitate in the chloroform layer was removed by filtration. Then, the chloroform layer was discarded, and three washing cycles with chloroform (50 ml×3) were performed to remove traces of 8-hydroxyquinoline. The water layer was concentrated to 50 ml and a white-color ε-Acryloyl L-Lysine was precipitated upon addition of tetrahydrofuran. The procedure is illustrated in Scheme 5.1. The yield was 4.1 g (93%). This structure was confirmed by .sup.1HNMR (D.sub.2O, d ppm): 1.43 (m, 2H, CH.sub.2), 1.61 (m, 2H, CH.sub.2), 1.84 (m, 2H, CH.sub.2), 3.25 (t, 2H, CH.sub.2), 3.75 (t, 2H, CH.sub.2), 5.70 (d, 1H, CH.sub.2═CH[trans]), 6.19 (d, 1H, CH.sub.2═CH[cis]), 6.21 (dd, 1H, CH.sub.2═CH).
(113) ##STR00006##
(114) Synthesis of Poly(ε-acryloyl-L-lysine):
(115) The initiator 4,4-azobis-4-cyano valeric acid (12.5 mg, 45×10.sup.−3 mmol=0.4 mol % with respect to monomer) was dissolved in water (40 mL) and added to the monomer (2.5 g, 0.0125 mmol). Then, the reaction mixture was stirred at room temperature and the pH was adjusted to a value of about 6 to 7. Then, the resulting mixture was degassed by five freeze-pump cycles and subsequently polymerized at 75° C. for 24 h. The reaction was monitored using HNMR and, at 100% conversion, the polymer was precipitated using tetrahydrofuran and acetone. The procedure is illustrated in Scheme 5.2 and generally follows the procedure of Weller (2013). After vacuum-drying, a colorless solid was obtained and yield was 2.5 g (100%). This structure was confirmed by .sup.1HNMR (D.sub.2O, d ppm): 1.26-1.30 (br, 2H, CH2), 1.35-139 (br, 2H, CH2), 1.54 (br, 2H, CH2), 1.76 (br, 2H, CH2), 1.99 (br, 1H, CH), 2.99 (br, 2H, CH2), 3.71 (bm, 1H, CH), 7.88 (br, 1H, CONH).
(116) ##STR00007##
(117) Synthesis of poly(2,2-(5-acylamido-1-carboxypentylazanediyl) diacetic acid), poly(NTA-Am-100):
(118) Under a nitrogen (N.sub.2) atmosphere, bromoacetic acid (12.5 g, 0.09 mol), NaOH (3.5 g, 0.09 mol) and 50 ml of water were added to a two-neck round-bottomed flask, and the mixture was stirred at room temperature for 10 min. This solution was added drop-wise with stirring to an aqueous solution (100 mL) containing poly(ε-acryloyl-L-lysine) (2.5 g, 0.0125 mol) at 50° C. The reaction mixture was kept at 50° C. for 24 h with occasional addition of 30% NaOH to maintain the pH at 10.0. Then the pH was adjusted to a value of 1.0 by adding concentrated HCl. The supernatant was decanted, the remaining precipitate was dissolved by addition of 30% NaOH, and the solution was again adjusted to pH 1.0 with concentrated HCl. This process was repeated 2 times, and the precipitate was filtered and dried in vacuo for 12 h. The procedure is illustrated in Scheme 5.3. The resulting white poly(2,2-(5-acrylamido-1-carboxypentylazanediyl) diacetic acid) solid, 2.8 g (70% yield, degree of functionalization is 81%). This structure was confirmed by .sup.1HNMR (D.sub.2O, d ppm): 1.33 (bm, 2H, CH2), 1.43 (br, 2H, CH2), 1.53 (br, 2H, CH2), 1.78 (br, 2H, CH2), 2.0 (br, 1H, CH), 3.0 (br, 2H, CH2), 3.49 (br, 1H, CH), 3.59 (br, 4H, 2×CH2), 7.86 (br, 1H, CONH).
(119) ##STR00008##
(120) Synthesis of Poly(ε-acryloyl-L-lysine-co-acrylic acid), poly(Lys-50-co-AA-50):
(121) As described above, the initiator 4,4-azobis-4-cyano valeric acid (12.5 mg, 45×10.sup.−3 mmol=0.4 mol % with respect to monomer) was dissolved in water (40 mL) and added to the monomers, c-Acryloyl L-Lysine (1.25 g, 0.00625 mmol) and acrylic acid (0.44 g, 0.00625 mmol) (i.e., a 50:50 molar monomer ratio of Lys:AA). Then, the reaction mixture was stirred at room temperature for 10 min and the pH was adjusted to a value of about 6 to 7. Then, the mixture was degassed by five freeze-pump cycles and subsequently polymerized at 75° C. for 24 h. The reaction was monitored using HNMR. The polymer was precipitated using tetrahydrofuran and acetone. The procedure is illustrated in Scheme 5.4. After vacuum drying a colorless solid was obtained and yield was 1.38 g (82%, Only 77% of lysines are incorporated in to the polymer). This structure was confirmed by .sup.1HNMR (D.sub.2O, d ppm): 1.28 (br, 2H, CH2), 1.40 (br, 2H, CH2), 1.47-1.71 (br, 4H, 2×CH2), 1.76 (br, 2H, CH2), 1.90 (br, 2H, 2×CH), 2.44 (NH2), 2.99 (br, 2H, CH2), 3.41-3.48 (m, 1H, CH—NH2), 3.59 (br, 1H, CH—NH3+), 7.82 (br, 1H, CONH).
(122) ##STR00009##
(123) Synthesis of Poly(2,2-(5-acrylamido-1-carboxypentylazanediyl) diacetic acid-co-acrylic acid), poly(NTA-Am-50-co-AA-50):
(124) Under a nitrogen (N.sub.2) atmosphere, bromoacetic acid (4.7 g, 0.034 mol), NaOH (1.35 g, 0.034 mol), and 50 ml of water were added to a two-neck round-bottomed flask, and the mixture was stirred at room temperature for 10 min. This solution was added drop-wise with stirring to an aqueous solution (50 mL) containing poly(Lys-50-co-AA-50) (1.38 g, 0.0048 mol of lysine repeating units) at 50° C. The reaction mixture was kept at 50° C. for 24 h with occasional addition of 30% NaOH to maintain the pH at 10.0. Then the pH was adjusted to a value of 1.0 by adding concentrated HCl. The supernatant was decanted, the remaining precipitate was dissolved by addition of 30% NaOH, and the solution was again adjusted to pH 1.0 with concentrated HCl. This process was repeated 2 times, and the precipitate was filtered and dried in vacuo for 12 h. The procedure is illustrated in Scheme 5.5. The resulting was white poly(2,2-(5-acrylamido-1-carboxypentylazanediyl) diacetic acid-co-acrylic acid) solid, 0.80 g (degree of functionalization is 87%). This structure was confirmed by .sup.1HNMR (D.sub.2O, d ppm): 1.31 (br, 2H, CH2), 1.41 (br, 2H, CH2), 1.53 (br, 4H, 2×CH2), 1.67-1.75 (br, 2H, CH2), 1.87-1.96 (br, 2H, 2×CH), 2.99 (br, 2H, CH2), 3.58 (br, 5H, 2×CH2 and CH), 7.85 (br, 1H, CONH).
(125) ##STR00010##
(126) Synthesis of Poly(ε-acryloyl-L-lysine-co-acrylic acid), poly(Lys-25-co-AA-75):
(127) As described above, the initiator 4,4-azobis-4-cyano valeric acid (18.9 mg, 45×10.sup.−3 mmol=0.4 mol % with respect to monomer) was dissolved in water (40 mL) and added to the monomers, ε-Acryloyl L-Lysine (0.945 g, 0.0047 mmol) and acrylic acid (1.0 g, 0.0142 mmol) (i.e., a 25:75 molar monomer ratio of Lys:AA. Then, the reaction mixture was stirred at room temperature for 10 min and pH was adjusted to a value of about 6 to 7. Then the mixture was degassed by five freeze-pump cycles and subsequently polymerized at 75° C. for 24 h. The reaction was monitored using HNMR. At the end, the polymer was precipitated using tetrahydrofuran and acetone. After vacuum drying a colorless solid was obtained and yield was 1.12 g (60%, Only 70% of lysines are incorporated in to the polymer). This structure was confirmed by .sup.1HNMR (D.sub.2O, d ppm): 1.29 (br, 2H, CH2), 1.40 (br, 2H, CH2), 1.47-1.70 (br, 4H, 2×CH2), 1.71-1.78 (br, 2H, CH2), 1.96 (br, 2H, 2×CH), 2.45 (NH2), 3.02 (br, 2H, CH2), 3.41-3.48 (m, 1H, CH—NH2), 3.60 (br, 1H, CH—NH3+), 7.81 (br, 1H, CONH).
(128) Synthesis of Poly(2,2-(5-acrylamido-1-carboxypentylazanediyl) diacetic acid-co-acrylic acid), poly(NTA-Am-25-co-AA-75):
(129) Under a nitrogen (N.sub.2) atmosphere, bromoacetic acid (4.7 g, 0.034 mol), NaOH (1.35 g, 0.034 mol) and 50 ml of water were added to a two-neck round-bottomed flask, and the mixture was stirred at room temperature for 10 min. This solution was added drop-wise with stirring to an aqueous solution (50 mL) containing poly(Lys-25-co-AA-75) (1.12 g) at 50° C. The reaction mixture was kept at 50° C. for 24 h with occasional addition of 30% NaOH to maintain the pH at 10.0. Then the pH was adjusted to a value of 1.0 by adding concentrated HCl. The supernatant was decanted, the remaining precipitate was dissolved by addition of 30% NaOH, and the solution was again adjusted to pH 1.0 with concentrated HCl. This process was repeated 2 times, and the precipitate was filtered and dried in vacuo for 12 h. The resulting white poly(2,2-(5-acrylamido-1-carboxypentylazanediyl) diacetic acid-co-acrylic acid) solid, 1.46 g (degree of functionalization is 81%). This structure was confirmed by 1HNMR (D.sub.2O, d ppm): 1.32 (br, 2H, CH2), 1.42 (br, 2H, CH2), 1.50-1.52 (br, 4H, 2×CH2), 1.67-1.73 (br, 2H, CH2), 1.90-1.97 (br, 2H, 2×CH), 3.0 (br, 2H, CH2), 3.55 (br, 5H, 2×CH2 and CH), 7.83 (br, 1H, CONH).
Example 6: Synthesis of Star Polyacid Polymers
(130) The following example describes the synthesis of star polymers, in particular star polyacid polymers having free acid groups according to the disclosure.
Synthesis of four-armed star-poly(tert-butyl acrylate)
(131) Tert-butyl acrylate (tBA) (29 ml, 0.2 mol) and N,N,N′,N′,N″-pentamethyldiethylenetriamine (PMDETA) (0.05 ml, 0.25 mmol) were dissolved in acetone (10% v/v) in a 100 ml Schlenk flask and the solution mixture was degassed by five freeze-thaw cycles. After three freeze-thaw cycles, the mixture was frozen under an N.sub.2 atmosphere, CuBr (36 mg, 0.25 mmol) and tetra (2-bromoisobutyryl) pentaerythritol (0.3 g, 0.5 mmol) were added, and the mixture was degassed by two freeze-thaw cycles. The mixture was stirred over 24 h at 60° C. The polymer was then precipitated using distilled water-methanol mixture, and the resulting white-color poly(tert-butyl acrylate) was dried in vacuo. The star-poly(tert-butyl acrylate) was characterized by .sup.1HNMR, gel permeation chromatography (GPC). Molecular weight (M.sub.n, GPC) and molecular weight distribution were also determined. Similar to this procedure, star polymers with 3 and 6 arms were synthesized.
Synthesis of four arms star-poly(acrylic acid) [Star-PAA-4]
(132) Star-poly(tert-butyl acrylate) (2.3 g) was dissolved in 20 ml dichloromethane. To this reaction mixture, trifluoroacetic acid (10 g) was added carefully. After 24 h of reaction time, the precipitated star poly(acrylic acid) was recovered and freeze dried (2.0 g). This procedure is shown in Scheme 6.1 for a four-armed star polyacid polymers, and structures of analogous three- and six-armed polymers are also shown.
(133) ##STR00011## ##STR00012##
(134) Synthesis of four arm star-poly(2-(dimethylamino)ethyl methacrylate) polycation [Star-(PDMAEMA-4)]:
(135) Similar to the previous procedure, N,N-dimethylaminoethyl methacrylate (DMAEMA) (25 ml, 0.15 mmol) and N,N,N′,N″,N′″,N′″-hexamethyltriethylenetetraamine (HMTETA) (0.27 ml, 1.0 mmol) were dissolved in THF (10 ml) in a 100 ml Schlenk flask and the solution mixture was degassed by five freeze-thaw cycles. After three freeze-thaw cycles, mixture was frozen under an N.sub.2 atmosphere, CuBr (72 mg, 0.5 mmol) and tetra (2-bromoisobutyryl) pentaerythritol (0.3 g, 0.5 mmol) were added and mixture was degassed by two freeze-thaw cycles. The mixture was stirred over 24 h at room temperature. The polymer was then precipitated using a heptane-ethylacetate mixture, and the resulting white-color star-poly(2-(dimethylamino)ethyl methacrylate) was dried in vacuo. The procedure is illustrated in Scheme 6.2. The star-poly(2-(dimethylamino)ethyl methacrylate) was characterized by .sup.1HNMR, gel permeation chromatography (GPC). Molecular weight (M.sub.n, GPC) and molecular weight distribution were also determined. Similar to this procedure, star polymers with 3 and 6 arms were synthesized.
(136) ##STR00013## ##STR00014##
Example 7: Synthesis of Polyethylene Oxide-Based Polyacid Polymers
(137) The following example describes the synthesis of polyacid polymers having pendent metal binding ligands according to the disclosure, in particular those having a polyethylene oxide polymer backbone and pendent nitrilotriacetic free acid groups as metal binding ligands.
(138) Synthesis of poly(epichlorohydrin) backbone:
(139) Synthesis of poly(epichlorohydrin) poly(EPCH) was carried out according to the procedure of Carlotti (2008) with some modifications (Scheme 7.1). First, dry epichlorohydrin (8.64 ml, 0.11 mol, [EPCH]=3M) was dissolved in dry Toluene (23 ml) in 200 ml Schlenk flask. To that reaction mixture initiator NOct.sub.4Br (0.17 g, 0.31 mmol) was added. The reaction was stirred under N.sub.2 for 5 min and three freeze-thaw cycles were performed. Then reaction mixture was cooled with liquid nitrogen and catalyst i-Bu.sub.3Al (1.72 ml, 1.7 mmol) added using a syringe. Conversion was monitored using .sup.1HNMR. At the end of the reaction, few drops of ethanol were added to quench the reaction. Toluene was removed by rotavap and resulted polymer was washed with 3% V/V HCl in ethanol. Then the final product was dried under vacuum. The yield of resulted product was 9.0 g (88%). This product was characterized using .sup.1HNMR (CDCl.sub.3, d ppm), 3.58-3.67 (br, 1H), 3.69-3.79 (br, 4H).
(140) ##STR00015##
Synthesis of poly(epichlorohydrin-co-glycidyl methoxyethoxyethoxy-oxirane) backbone [poly(EPCH-co-GMEEO)]
(141) Poly(EPCH-co-GMEEO) can be synthesized similarly to the procedure described above. Epichlorohydrin and 2-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)oxirane can be copolymerized under the condition shown in Scheme 7.2 to get the desired polymer backbone.
(142) ##STR00016##
Synthesis of poly(glycidyl-N,N bis-(carboxymethyl)-L-Lysine) [poly(GNTA)]
(143) Poly(GNTA) can be synthesized as shown the scheme by reacting poly(epichlorohydrin) with N,N bis-(carboxymethyl)-L-Lysine (Aminobutyl NTA) under basic condition (Scheme 7.3).
(144) ##STR00017##
(145) Synthesis poly(glycidyl amine) [poly(GAm)]: Poly(GAm) can be synthesized according to the procedure of Meyer (2011), which is essentially a two-step synthesis. First, poly(glycidyl azide) (poly(GAz)) is synthesized as shown in Scheme 7.4. The resulting poly(GAz) polymer is then reacted with triphenyl-phosphine and water to get the final product poly(GAm).
(146) ##STR00018##
(147) Synthesis of poly(glycidyl-N,N bis-(carboxymethyl)-L-Lysine-co-glycidyl methoxyethoxyethoxyoxirane) [poly(GNTA-co-GMEEO)]:
(148) Poly(GNTA-co-GMEEO) can be synthesized as shown Scheme 7.5 by reacting poly(EPCH-co-GMEEO) with N,N bis-(carboxymethyl)-L-Lysine (Aminobutyl NTA) under basic conditions.
(149) ##STR00019##
(150) Synthesis of poly(glycidyl amine-co-glycidyl methoxyethoxyethoxyoxirane) [poly(GAm-co-GMEEO)]:
(151) Poly(GAm-co-GMEEO) can be synthesized similarly to the procedure described above. First, a corresponding azide intermediate can be synthesized by reacting poly(EPCH-co-GMEEO) and sodium azide under the conditions shown in Scheme 7.6. Then, the resulting poly(GAz-co-GMEEO) can be reacted under the conditions shown in the scheme to obtain the poly(GAm-co-GMEEO) product.
(152) ##STR00020##
Example 8: PNTA-Functionalized Membranes
(153) This example describes a convenient synthesis of nitrilotriacetate (NTA)-containing polymers and subsequent layer-by-layer adsorption of these polymers in membrane pores. The resulting films form NTA-metal-ion complexes isolate multilayers of polyhistidine-tagged proteins that bind to the metal-ion complexes. Moreover, adsorption of films in porous nylon membranes gives materials that capture 45 mg of His-tagged ubiquitin per cm.sup.3. However, the binding capacity decreases with the protein molecular weight. Due to the high affinity of NTA for metal ions, these membranes show modest leaching of Ni.sup.2 in binding and rinsing buffers. Adsorption of NTA-containing polymers is a simple method to create metal- and protein-binding films and may facilitate development of disposable membranes that rapidly purify tagged proteins.
(154) Synthesis of poly(2,2-(5-acrylamido-1-carboxypentylazanediyl) diacetic acid) [PNTA-100 or PNTA], an NTA-containing polymer, and incorporation of this polymer into polyelectrolyte multilayers in functionalized porous membranes 104 to capture metal ions as well as proteins that bind to these immobilized ions is described (
(155) Materials:
(156) Poly(allylamine hydrochloride) (PAH, M.sub.w=120,000-210,000, Alfa-Aesar), branched polyethyleneimine (BPEI, M.sub.w=25,000, Sigma-Aldrich), and poly(acrylic acid) (PAA, M.sub.w=90,000, 25% aqueous solution, Polysciences) were employed for LBL deposition. Hydroxylated nylon membranes (LOPRODYNE LP, Pall, 1.2 μm pore size, 110 μm thick) were cut into 25 mm-diameter discs prior to use. Synthesis and characterization of PNTA-100, PNTA-44 and PNTA-19 are described below. N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), Na, No-bis(carboxymethyl)-L-lysine hydrate (aminobutyl NTA), 3-mercaptopropionic acid (MPA, 99%), L-lysine monohydrochloride (98%), acryloyl chloride (97%, contains <210 ppm MEHQ as stabilizer), copper(II) carbonate basic (≥95%), 8-hydroxyquinoline and bromoacetic acid (≥97%) were purchased from Sigma-Aldrich. Coomassie protein assay reagent (Thermo Scientific), Histidinee-tagged ubiquitin (HisU, human recombinant, Enzo Life Sciences), and conconavalin A (Sigma-Aldrich, Con A) from Canavaliaensiformis (Jack bean) were used as received. Phenylalanine amminomutase (PaPAM) and L-threonine aldolase were prepared as described previously. Aqueous solutions containing 1 mg/mL of PAH or 1 mg/mL of PNTA-X were prepared in deionized water (18.2 MΩcm, Milli-Q). PNTA-containing solutions were obtained by first dissolving the polymer with the addition of 6 M NaOH to achieve a pH 9.0 solution and when desired adjusting the pH to 3.0 with 6 M HCl.
(157) Membrane Modification with (PAH/PNTA-X) Films:
(158) With reference to
(159) Metal-Ion Binding and Leaching in (PAH/PNTA-X)-Modified Membranes:
(160) After membrane modification with polyelectrolyte multilayers, 0.1 M CuSO.sub.4 or NiSO.sub.4 solutions were circulated through the membrane for 1 h, followed by a 20-mL water wash. The bound metal ions were eluted with two 5-mL aliquots of 0.1 M EDTA (pH 7.6) or 2% HNO.sub.3, and the concentrations of metal ions in these solutions were determined using atomic absorption spectroscopy with calibration curves. Standard solutions contained 0 to 10 ppm metal ions in 0.1 M EDTA or in 2% HNO.sub.3. The metal-ion binding capacity was calculated by dividing the mass of eluted metal ion by the volume of membrane, which is about 0.035 cm.sup.3 (the membranes are 110 μm thick). A GE Healthcare HITRAP IMAC FF column was used as a comparison and loaded with Ni.sup.2+ by passing 2 mL of 0.1 M NiSO.sub.4 through the syringe column (flow rate of 1 mL/min) followed by 75 mL of deionized water. To study metal-ion leaching, Cu.sup.2+ leaching values from membranes modified with (PAH/PNTA-100).sub.2 and PAA/BPEI/PAA-NTA were compared. After Cu.sup.2+ loading and rinsing with water, the membranes were washed sequentially with 2.5 mL (70-75 membrane bed volumes) of four different buffers (pH 7.4) and then 0.1 M EDTA. This protocol was also followed to determine the amount of Ni.sup.2+ binding/leaching in (PAH/PNTA-100).sub.2- or PAA/BPEI/PAA-NTA-modified membranes. For comparison, a GE Healthcare HITRAP IMAC FF column (1 mL) was washed with 75 bed volumes (75 mL) of the same buffers (
(161) His-tagged Protein Binding in Nylon Membranes: Functionalized membranes 104 with an exposed diameter of 1 cm were used for all protein-binding studies. Solutions for His-tagged protein P binding to Ni.sup.2+-containing membranes 104 included 0.3 mg of protein P per mL in 20 mM phosphate buffer at pH 7.4. The membranes 104 were loaded with buffered protein P (
Synthesis of Homo-Polymer PNTA-100 and Co-Polymers PNTA-44 and PNTA-19
(162) This example includes a synthesis of a polymer that contains metal-ion-binding NTA groups. Other syntheses of NTA-containing polymers derivatize a polymer with aminobutyl NTA, which is expensive to purchase or prepare due to protection and deprotection steps. Schemes 5.1 and 5.2 above outline the synthesis of poly(2,2-(5-acrylamido-1-carboxypentylazanediyl) diacetic acid) [PNTA-100]. The strategy includes a modified literature procedure to prepare ε-acryloyl L-lysine. The Cu.sup.2+-protection strategy in this monomer synthesis bypasses the cumbersome protection/deprotection steps in other protocols. Also, the procedure avoids lengthy column purification. Subsequent initiation of free radical polymerization with 4,4′-azobis(4-cyanovaleric acid) yields the intermediate polymer, poly(ε-acryloyl L-lysine) [PLys-100], with 92% conversion, and the product .sup.1H NMR spectrum is consistent with polymerization. Based on .sup.1H NMR end-group analysis, the PLys-100 has an average degree of polymerization (DP.sub.n) of 340, which is close to the monomer-to-initiator ratio of 280. Finally, carboxymethylation of PLys-100 using bromoacetic acid leads to PNTA-100 with 70% yield, and the .sup.1H NMR spectrum of the product confirms the formation of the desired polymer. Integration of the spectrum signals suggests addition of 1.6 carboxymethyl groups per repeat unit of PLys-100, and elemental analysis implies slightly greater derivatization.
(163) Relatively low aqueous swelling of (PAH/PNTA-100).sub.n films containing bound metal ions may limit protein access to metal-ion complexes. Thus, to increase swelling, acrylic acid (AA) comonomers are incorporated into PNTA. Deprotonation of the AA repeat units after film formation should increase swelling in water to facilitate protein capture. The synthesis of these materials includes copolymerization of AA and ε-acryloyl L-lysine and reaction of the resulting polymers with 2-bromoacetic acid. Specifically, poly(NTA-co-AA) was prepared aiming to achieve polymers with 25% or 50% of the repeating units containing NTA ligands along with the corresponding 75% or 50% AA units, respectively. NMR analysis suggests that these polymers have about 19% and 44% NTA-containing units, respectively, so they are denoted as PNTA-19 and PNTA-44.
(164) LBL Deposition of (PAH/PNTA-X).sub.n Films:
(165) This example creates thin films that selectively bind metal ions and proteins and to tune the binding properties of these films through varying composition and deposition conditions. Alternating adsorption of PAH and PNTA-X is a simple technique for preparing films with metal-ion-binding groups, and because PAH and PNTA-X are weak polyelectrolytes, the deposition pH will affect the film thickness and structure. The pH of the polyelectrolyte solution controls the degree of protonation and hence the charge density of the polymer, which influences both the polymer conformation and the degree of ionic cross-linking in layer-by-layer films. In aqueous solutions, the pK.sub.a values of free NTA (analog of the metal-binding group in PNTA-100 repeating units) are around 9.7 for the ammonium group and below 3 for the —COOH groups. Titration of PNTA-100 with 0.1 M HCl shows the presence of fully protonated tertiary amines in the polymer below pH 9, whereas the three carboxylic acid groups protonate below pH 3. In addition, on going from pH 3.0 to 9.0, the fraction of protonated amines in PAH decreases from 96 to 30%.
(166) LBL adsorption of (PAH/PNTA-100).sub.5 films at pH 3 yields a thickness of ˜20 nm. Deposition at pH 9 also gives a thickness of 20 nm for these films. In contrast, adsorption of PNTA-100 at pH 3 and PAH at pH 9 leads to much greater thicknesses. Ellipsometry and AFM data indicate that such (PAH/PNTA-100).sub.5 films are 400-500 nm thick. At pH 9, PAH likely adsorbs with a significant degree of deprotonation, and protonation of the adsorbed PAH at pH 3 during PNTA-100 deposition leads to excess positive surface charge that enhances PNTA-100 adsorption. Subsequent deprotonation of the adsorbed PNTA-100 likely increases the net negative charge density in the film and augments PAH deposition at pH 9. Surprisingly, addition of NaCl to deposition solutions decreases film thickness significantly (see
(167) Although adsorption of PNTA-100 at pH 3 and PAH at pH 9 without added supporting electrolyte leads to thick coatings, these films show low protein binding, which may suggest significant electrostatic cross-linking. In contrast films prepared with the same deposition pH values from solutions containing 0.5 M NaCl show multilayer protein binding. Thus, adsorption of (PAH/PNTA-X).sub.n films from polymer solutions containing 0.5 M NaCl is a focus of the following results. (PAH/PNTA-44).sub.5 and (PAH/PNTA-19).sub.5 films are thicker than the corresponding (PAH/PNTA-100).sub.5 coatings when both films are deposited from solutions containing 0.5 M NaCl (adsorption at pH 9 for PAH and pH 3 for PNTA-X). Because PNTA-44-containing polymer films bind approximately the same amount of protein as PNTA-100-containing films, hereafter only films containing PNTA-100 and PNTA-19 are compared.
(168) Metal-Ion Binding and Leaching from Membranes Modified with NTA-Containing Films:
(169) The composition of the initial polyelectrolyte adsorbed to a membrane is important to create a stable, charged surface for further film growth, and PAA adsorbs strongly to nylon membranes. Thus, a membrane modification with PAA/PAH/PNTA-X films was used. Adsorption of PAA/PAH/PNTA-100 does not cause major changes to the membrane morphology, whereas deposition of a PAA(PAH/PNTA-100).sub.2 film begins to decrease porosity. Swelling in buffer will further decrease porosity and limit flow.
(170) Metal-Ion-Binding Capacities for Several Modified Membranes:
(171) For both Cu.sup.2+ and Ni.sup.2+, the binding capacities more than double on going from PAA/PAH/PNTA-100 to PAA/(PAH/PNTA-100).sub.2 films. Notably, the Cu.sup.2+ binding capacity is 60-80% higher than that for Ni.sup.2+ (a membrane with a PAA/(PAH/PNTA-100).sub.2 film binds 8.9 mg of Ni.sup.2+ per cm.sup.3). Because free —COOH groups in PAA likely bind some Cu.sup.2+, the Cu.sup.2+ capture by membranes modified with a PAA/PAH film was determined. Such membranes bind only 1.7 mg of Cu.sup.2+ per cm.sup.3 of membrane, which is not sufficient to account for the difference in Cu.sup.2+ and Ni.sup.2+ binding. Thus the higher Cu.sup.2+ binding capacity likely reflects stronger affinity for NTA sites in the film. Because of electrostatic interactions between NTA and protonated amines, the NTA ligands may exhibit a range of affinities for metal ions, and low affinity sites may not capture Ni.sup.2+ under the loading conditions.
(172) In addition to metal-ion-binding capacity, leaching of bound metal ions may also affect protein purification. Initially, leaching values of Ni.sup.2+ from PAA/BPEI/PAA-NTA- and PAA(PAH/PNTA-100).sub.2-modified membranes were compared. In the PAA/BPEI/PAA-NTA-modified membrane, the binding buffer, which contains only 10 mM imidazole, elutes about 50% of the bound Ni.sup.2+ (
(173) The PNTA-100-containing films show much less leaching than membranes with PAA/BPEI/PAA-NTA (
(174) Protein Binding to Cu.sup.2+/Ni.sup.2+-containing Membranes: Based on experiments with polyelectrolyte multilayers on Au-coated wafers, (PAH/PNTA-19).sub.n binds more protein than (PAH/PNTA-100).sub.n. However, passage of a 0.3 mg/mL Con A solution through membranes modified with either PAA/PAH/PNTA-100-Cu.sup.2+ or PAA/PAH/PNTA-19-Cu.sup.2+ gave similar binding capacities of only 20-25 mg of Con A per cm.sup.3 of membrane. Thus the acrylic acid groups in PNTA-19 did not increase protein binding in membranes containing only one PAH/PNTA-X—Cu.sup.2+ bilayer. The effect of the additional acrylic acid groups on protein binding may prove more important in PAA(PAH/PNTA-19).sub.n—Cu.sup.2+ films, but adsorption of a second PAH/PNTA-19 bilayer plugged membrane pores.
(175) Because PAA(PAH/PNTA-100).sub.2 films do not plug membrane pores, binding of HisU to membranes modified with PAA(PAH/PNTA-100).sub.2-Ni.sup.2+ coatings was investigated. The binding capacity of these membranes is 18±1 mg/mL. Interestingly, for a PAA/PAH/PNTA-100-Ni.sup.2+-modified membrane, the His-U binding capacity is 47±5 mg/mL based on breakthrough curves, and elution gives a binding capacity of 40±3 mg/mL. Adsorption of a second PAH/PNTA-100 bilayer leads to less protein capture. Plugging of pores may block some binding sites, and the SEM investigation suggests that highly water-swollen (PAA/PAH/PNTA-100).sub.2-Ni.sup.2+ films may block pores.
(176) Membranes containing PAA/PAH/PNTA-19-Ni.sup.2+ bind the same amount of protein (46 mg of His U per mL) as membranes with PAA/PAH/PNTA-100-Ni.sup.2+. Thus, the acrylic acid groups in PNTA-19 again do not enhance binding. As Table 1 shows, membranes with PAA/BPEI/PAA-NTA films bind 89 mg of His U/mL of membrane, or approximately twice the amount of His U captured in membranes modified with PAA/PAH/PNTA-19-Ni.sup.2+ or PAA/PAH/PNTA-100-Ni.sup.2+. The PAA/BPEI/PAA-NTA-Ni.sup.2+ films apparently present the most protein-accessible Ni.sup.2+ binding sites.
(177) Finally, proteins were used with four different molecular masses (Table 1, bottom two rows) to study the effect of the protein size on the binding capacity of modified membranes. PAA/PAH/PNTA-19 coatings were examined, because on a flat substrate films containing PNTA-19 exhibit binding that varies with molecular mass. Both PAA/BPEI/PAA-NTA- and PAA/PAH/PNTA-19-modified membranes show their highest binding capacities of 89 and 46 mg/ml respectively, with the smallest protein, His U. Unfortunately, the binding drops to around 10 mg/mL for His-tagged PaPAM which has a molecular weight of 59 kDa. Nevertheless, we do not know if this trend is protein-dependent. In most cases membranes with PAA/PAH/PNTA-19 bind around ⅓ to ½ of the protein captured in membranes with PAA/BPEI/PAA-NTA. However, even the capacity of PAA/PAH/PNTA-modified membranes is comparable to that of commercial beads. Moreover, this new modification strategy is easy to apply and minimizes metal-ion leaching compared to membranes with PAA/BPEI/PAA-NTA.
(178) TABLE-US-00001 TABLE 1 Protein-binding capacities of membranes modified with PAA/BPEI/PAA-NTA-Ni.sup.2+ and PAA/PAH/PNTA-X-Ni.sup.2+ films. Binding capacity (mg/mL).sup.a His U Con A Aldolase PaPAM From From From From break break break break Membrane through From through From through From through From modification curve elution curve elution curve elution curve elution PAA/PAH/PNTA-100 47 40 25 28 — — — — PAA(PAH/PNTA-100).sub.2 — — 33 43 — — — — PAA/PAH/PNTA-19 46 34 25 30 22 18 7.8 — PAA/BPEI/PAA-NTA 89 85 73 70 55 56 11 14
(179) Summary: This example shows a simple approach, LBL adsorption with polymers containing NTA groups, to create films for strong metal-ion binding and selective capture of His-tagged proteins. Reaction of poly(ε-acryloyl L-lysine) with chloroacetic acid provides a convenient route to NTA-containing polymers, and adsorption of a PAA/PAH/PNTA-100-Ni.sup.2+ film in a porous membrane yields a His-tagged ubiquitin binding capacity of 47±5 mg/mL, which is comparable to the capacity of commercial beads. Films with PNTA-100 show less metal-ion leaching than coatings containing PAA derivatized with aminobutyl NTA, probably because weak binding to residual acid groups of PAA acid promotes leaching. The His-tagged protein-binding capacity of (PAH/PNTA-X)-Ni.sup.2+-modified membranes is half of that for membranes modified through adsorption of PAA/BPEI/PAA followed by aminobutyl NTA derivatization. However, direct adsorption of PAH and PNTA-X in membranes is simpler than previous membrane modification methods and may lead to inexpensive, disposable membranes for rapid purification of His-tagged protein. .sup.aProtein binding occurred from solutions containing 0.3 mg of protein per mL in pH 7.4 buffer, or pH 6.0 buffer for Con A. The Con A binding employed Cu.sup.2+ complexes rather than Ni.sup.2+. These experiments were performed only once.
(180) Because other modifications and changes varied to fit particular operating requirements and environments will be apparent to those skilled in the art, the disclosure is not considered limited to the example chosen for purposes of illustration, and covers all changes and modifications which do not constitute departures from the true spirit and scope of this disclosure.
(181) Accordingly, the foregoing description is given for clearness of understanding only, and no unnecessary limitations should be understood therefrom, as modifications within the scope of the disclosure may be apparent to those having ordinary skill in the art.
(182) All patents, patent applications, government publications, government regulations, and literature references cited in this specification are hereby incorporated herein by reference in their entirety. In case of conflict, the present description, including definitions, will control.
(183) Throughout the specification, where the articles, compositions, processes, kits, or apparatus are described as including components, steps, or materials, it is contemplated that the compositions, processes, or apparatus can also comprise, consist essentially of, or consist of, any combination of the recited components or materials, unless described otherwise. Component concentrations can be expressed in terms of weight concentrations, unless specifically indicated otherwise. Combinations of components are contemplated to include homogeneous and/or heterogeneous mixtures, as would be understood by a person of ordinary skill in the art in view of the foregoing disclosure.
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