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Query: UMLS:C0267964 (PAA)
 2,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transgenic plants may prove to be one of the most economical systems for the large-scale production of proteins and peptides. Our goal is to develop an approach for protein recovery from canola that will be adaptable to a wide variety of recombinant proteins. For recombinant protein recovery, the two downstream processes considered were extraction of target protein and purification of recombinant protein from host proteins and other impurities. In these experiments, T4 lysozyme has been added to the canola extracts as an example protein to simulate recovery of recombinant proteins while evaluating the merits of several candidate precipitation strategies to understand selectivity behavior and how it might be affected by the presence of canola components. The ability of precipitating agents, such as acid and the polyelectrolytes Glass H and poly(acrylic acid) (PAA), was investigated. Approximately 70% of extracted canola proteins were precipitated by acid addition to pH 5, leaving about 90% of T4 lysozyme still in solution. Targeting T4 lysozyme for the precipitate phase by addition of oppositely charged polyelectrolyte was not successful in the presence of canola components. Addition of 2.5 times the PAA dosage required to precipitate pure T4 lysozyme to the spiked canola extract brought down only 40% of the T4 lysozyme. For the comparable result with Glass H, a 9 times higher dosage was required.
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PMID:Strategies for recombinant protein recovery from canolaby precipitation 1035 67

Adsorption chromatography in expanded beds is a widely used technology for direct capture of target proteins from fermentation broths. However, in many cases this method cannot be applied as a result of the strong tendency of cells or cell debris to interact with the adsorbent beads. To prevent contamination of the expanded bed with the biomass, STREAMLINE DEAE, anion exchanger designed for expanded bed adsorption, was modified with a layer of poly(acrylic acid) (PAA). The shielding layer of polyelectrolyte was attached to the surface of the matrix beads via electrostatic interactions. PAA with a high degree of polymerization was chosen to prevent diffusion of large polymer molecules into the pores of adsorbent. Thus, the shielding layer of PAA was adsorbed only at the mouth of the pores of STREAMLINE DEAE beads and only marginally decreased the binding capacity of the ion exchanger for bovine serum albumin, the model protein in this study. PAA-coated STREAMLINE DEAE practically did not interact with yeast cells, which otherwise bound strongly to the native adsorbent at neutral conditions. Cell-resistant PAA-coated anion exchanger was successfully used for isolation of BSA from the model protein mixture containing BSA, lysozyme (positively charged at applied conditions), and yeast cells. The layer of PAA was stable under mild elution conditions, and the modified adsorbent could be used in the repeated purification cycles.
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PMID:Polyelectrolyte-coated ion exchangers for cell-resistant expanded bed adsorption. 1215 16

Tobacco is widely used as a model plant for feasibility studies of recombinant protein production from transgenic plants. However, dealing with large quantities of biomass to recover recombinant proteins is a challenge for down-stream processing. In this study, the effect of isoelectric precipitation on native tobacco protein was first studied. Among the three acids studied, hydrochloric acid is shown to be more effective than acetic or citric acid, and at pH 4, 60% of native tobacco protein was precipitated by HCl. Egg white lysozyme was used as the model protein to test the feasibility of polyelectrolyte precipitation in protein recovery from tobacco extract. Precipitation of lysozyme at pH 7 was shown ineffective probably because of the interference of polyphenolic acids. However, after isoelectric precipitation at pH 5 poly(acrylic) acid (PAA) was shown to precipitate 85% of the soluble lysozyme when the polymer dosage was increased to 1.5 mg polymer/mg lysozyme, while negligible amounts of native tobacco protein was co-precipitated. Lysozyme precipitation by PAA in tobacco extract obtained at pH 5 was also studied, and lysozyme yield was significant improved.
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PMID:Lysozyme purification from tobacco extract by polyelectrolyte precipitation. 1584 88

The adsorption of two different proteins at a planar poly(acrylic acid) (PAA) brush was studied as a function of the ionic strength of the protein solutions applying total internal reflection fluorescence (TIRF) spectroscopy. Planar PAA brushes were prepared with a grafting density of 0.11 nm(-2) and were characterized using X-ray reflectometry. Hen egg-white lysozyme and bovine serum albumin (BSA) were used as model proteins, which have a net positive and negative charge at neutral pH-values, respectively. It has been found that both proteins adsorb strongly at a planar PAA brush at low ionic strength. Whereas lysozyme interacts with a PAA brush under electrostatic attraction at neutral pH-values, BSA binds under electrostatic repulsion at pH > 5. Even at pH = 8, significant amounts of BSA are adsorbed to a planar PAA brush. In addition, the reversibility of BSA adsorption has been characterized. Dilution of a BSA solution leads to an almost complete desorption of BSA from a PAA brush at short contact times. When the ionic strength of the protein solutions is increased to about 100-200 mM, a planar PAA brush appears largely protein-resistant, regardless of the protein net charge. The results of this study indicate that the salt-dependent protein affinity of a PAA brush represents a unique effect that must be explained by a novel protein-binding mechanism. On the basis of a recent model, it is suggested that a release of counterions is the most probable driving force for protein adsorption at a PAA brush. In a general view, this study characterizes a planar PAA brush as a new materials coating for the controlled immobilization of proteins whose use in biotechnological applications appears to be rewarding.
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PMID:Characterization of a planar poly(acrylic acid) brush as a materials coating for controlled protein immobilization. 1654 92

Polymeric coatings with high protein-binding capacities are important for increasing the output of affinity-based protein purification and decreasing the detection limits of antibody microarrays. This report describes the use of thick poly(acrylic acid) (PAA) brushes to immobilize as much as 80 monolayers of protein. The brushes were prepared using a recently developed procedure that allows polymerization of 100-nm-thick poly(tert-butyl acrylate) films from a surface in just 5 min along with hydrolysis of these films to PAA in 15 min. Covalent binding of bovine serum albumin (BSA) to PAA brushes that were activated using standard coupling agents, however, resulted in immobilization of less than two monolayers of BSA because of competitive hydrolysis of the esters in the activated film. In contrast, derivatization of PAA with nitrilotriacetate (NTA)-Cu2+ complexes yielded films capable of binding many monolayers of protein via metal-ion affinity interactions. For example, derivatization of 55-nm-thick PAA films with NTA-Cu2+ allowed immobilization of about 15 monolayers (5.8 microg/cm2 or 58 nm) of BSA. The binding capacity was even higher for myoglobin (7.7 microg/cm2) and anti-IgG (9.6 microg/cm2). Remarkably, electrostatic adsorption of lysozyme in 55-nm-thick, underivatized PAA resulted in as much as 80 monolayers (16.2 microg/cm2 or 162 nm) of adsorbed protein. Polymer synthesis, derivatization, and swelling, as well as BSA immobilization kinetics and thermodynamics were characterized using reflectance FT-IR spectroscopy, ellipsometry, and protein assays.
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PMID:High-capacity binding of proteins by poly(acrylic acid) brushes and their derivatives. 1661 75

This paper describes the preparation, characterization, and enzymatic activity of complex coacervate core micelles (C3Ms) composed of poly(acrylic acid) (PAA) and poly(N-methyl-2-vinyl pyridinium iodide)-b-poly(ethylene oxide) (PQ2VP-PEO) to which the antibacterial enzyme lysozyme is end-attached. C3Ms were prepared by polyelectrolyte complex formation between PAA and mixtures containing different ratios of aldehyde and hydroxyl end-functionalized PQ2VP-PEO. This resulted in the formation of C3Ms containing 0-40% (w/w) of the aldehyde end-functionalized PQ2VP-PEO block copolymer (PQ2VP-PEO-CHO). Chemical conjugation of lysozyme was achieved via reductive amination of the aldehyde groups, which are exposed at the surface of the C3M, with the amine groups present in the side chains of the lysine residues of the protein. Dynamic and static light scattering indicated that the conjugation of lysozyme to C3Ms prepared using 10 and 20% (w/w) PQ2VP-PEO-CHO resulted in the formation of unimicellar particles. Multimicellar aggregates, in contrast, were obtained when lysozyme was conjugated to C3Ms prepared using 30 or 40% (w/w) PQ2VP-PEO-CHO. The enzymatic activity of the unimicellar lysozyme-C3M conjugates toward the hydrolysis of the bacterial substrate Micrococcus lysodeikticus was comparable to that of free lysozyme. For the multimicellar particles, in contrast, significantly reduced enzymatic rates of hydrolysis, altered circular dichroism, and red-shifted tryptophan fluorescence spectra were measured. These results are attributed to the occlusion of lysozyme in the interior of the multimicellar conjugates.
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PMID:Complex coacervate core micelles with a lysozyme-modified corona. 1758 20

This study investigated the effect of ion-pairing of anionic polyelectrolytes: our novel poly(ethylene glycol)-block-oligo(vinyl sulfadimethoxine) (PEG-OVSDM) and poly(ethylene glycol)-block-poly(l-aspartic acid) (PEG-PAA) with cationic lysozyme on retention of protein stability during emulsification. Soluble lysozyme recovery after exposure to the deleterious interface was 42-88% (when ion-paired with PEG-OVSDM, PEG-OVSDM concentration dependent) compared to only 30% for free lysozyme. PEG-OVSDM provided a higher stabilization of lysozyme than PEG-PAA (36-60%). Lysozyme when recovered in the aqueous phase and analyzed by chromatography, enzymatic assay, fluorescence, and mass spectrometry showed no significant physicochemical change when compared with a lysozyme standard. Lysozyme was incorporated into poly(lactide-co-glycolide) (PLGA) microspheres via the typical double emulsion method. Incorporation of lysozyme complexes led to a higher encapsulation efficiency and loading amount, and a lower incidence of insoluble lysozyme aggregates compared to the control microspheres containing lysozyme only. More significantly, ion-pairing was able to dramatically reduce the initial lysozyme release to 18% compared with 50% from control microspheres and provided an overall better control of protein release. PEG-PAA was less effective than PEG-OVSDM in controlling the release probably due to weaker interactions between this polyelectrolyte and lysozyme. Manipulation of such polyelectrolyte-protein complexation may play a role in protein-controlled delivery.
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PMID:Role of a novel multifunctional excipient poly(ethylene glycol)-block-oligo(vinyl sulfadimethoxine) in controlled release of lysozyme from PLGA microspheres. 1839 74

The interaction of the proteins bovine serum albumin (BSA), lysozyme (Lys), lactoferrin (Lf), and fibronectin (Fn) with surfaces of protein-resistant poly(ethylene oxide) (PEO) and protein-adsorbing poly(acrylic acid) (PAA) fabricated by plasma-enhanced chemical vapor deposition has been studied with quartz crystal microbalance with dissipation monitoring (QCM-D). We focus on several parameters which are crucial for protein adsorption, i.e., the isoelectric point (pI) of the proteins, the pH of the solution, and the charge density of the sorbent surfaces, with the zeta-potential as a measure for the latter. The measurements reveal adsorption stages characterized by different segments in the plots of the dissipation vs frequency change. PEO remains protein-repellent for BSA, Lys, and Lf at pH 4-8.5, while weak adsorption of Fn was observed. On PAA, different stages of protein adsorption processes could be distinguished under most experimental conditions. BSA, Lys, Lf, and Fn generally exhibit a rapid initial adsorption phase on PAA, often followed by slower processes. The evaluation of the adsorption kinetics also reveals different adsorption stages, whereas the number of these stages does not always correspond to the structurally different phases as revealed by the D- f plots. The results presented here, together with information obtained in previous studies by other groups on the properties of these proteins and their interaction with surfaces, allow us to develop an adsorption scenario for each of these proteins, which takes into account electrostatic protein-surface and protein-protein interaction, but also the pH-dependent properties of the proteins, such as shape and exposure of specific domains.
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PMID:pH-dependent immobilization of proteins on surfaces functionalized by plasma-enhanced chemical vapor deposition of poly(acrylic acid)- and poly(ethylene oxide)-like films. 1854 95

Applying ATR-FTIR (attenuated total reflection Fourier transform infrared) and TIRF (total internal reflection fluorescence) spectroscopy, we have studied the secondary structure and aggregation properties of different proteins which are adsorbed at a poly(acrylic acid) (PAA) brush that covers a macroscopically large, planar surface. The PAA brush has been prepared on the surface of an ATR silicon crystal or a quartz plate. The preparation includes the deposition of a thin poly(styrene) film by spin-coating and the transfer of the diblock copolymer poly(styrene)-poly(acrylic acid) onto the hydrophobic film using the Langmuir-Schafer technique. It has been found that the proteins hen egg white lysozyme, bovine serum albumin, bovine alpha-lactalbumin, and bovine insulin adsorb spontaneously at a PAA brush at neutral pD values, albeit to different degrees. The secondary structure of the proteins was estimated from a decomposition of the amide I'-band in the observed ATR-FTIR spectra. Generally, the fractions of secondary structure elements recovered in this way were almost identical to those found when the proteins are native in solution. In addition, the tendency of insulin to form amyloid fibrils has also been tested when the protein is adsorbed at a planar PAA brush. Insulin is known to form amyloid fibrils in solution at low pH values and elevated temperatures. The experiments performed in this study suggest that a PAA brush does not promote fibril formation of insulin. Rather, insulin that is adsorbed at a PAA brush seems to be excluded from fibril formation pathways even at pD = 2 and 60 degrees C, where fibril formation of insulin is triggered in solution. Overall, the results of this study demonstrate that a planar PAA brush may serve as a mild environment for immobilized proteins.
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PMID:Native-like structure of proteins at a planar poly(acrylic acid) brush. 1909 23

In this study, the formation and disintegration of polyelectrolyte complex micelles is studied by dynamic light scattering titrations with the aim to assess the extent to which these complexes equilibrate. Also, the time evolution of samples at fixed (electroneutral) composition was followed to obtain information about the relaxation time of the complex formation. We find that, in 3.5 mM phosphate buffer with pH 7, polyelectrolyte complex micelles consisting of the positively charged homopolymer PDMAEMA(150), the negatively charged diblock copolymer PAA(42)-PAAm(417) (both having a pH-dependent charge), as well as the positively charged protein lysozyme slowly equilibrate with a relaxation time of about 2 days. The same structures were obtained, independent of the way the polymers and proteins had been mixed. In contrast, polyelectrolyte complex micelles (at the same pH) consisting of (pH-dependent) negatively charged homopolymer PAA(139), the pH-independent positively charged diblock copolymer P2MVP(41)-PEO(205), and the negatively charged protein alpha-lactalbumin did not equilibrate. The way in which solutions containing these macromolecules were mixed yielded different results that did not change over the period of at least a week.
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PMID:Reversibility and relaxation behavior of polyelectrolyte complex micelle formation. 1933 98


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