Loss Of Binding Capacity Research Articles

Bioinspired, Carbohydrate-Containing Polymers Efficiently and Reversibly Sequester Heavy Metals.

Water scarcity and heavy metal pollution are significant challenges in today's industrialized world. Conventional heavy metal remediation methods are often inefficient and energy-intensive, and produce chemical sludge. To address these issues, we developed a bioinspired, carbohydrate-containing polymer system for efficient and selective heavy metal removal. Using ring opening metathesis polymerization, we synthesized polymers bearing amphiphilic glucuronate side chains capable of selectively binding heavy metal cations in mixed media. In samples containing high concentrations of heavy metals (>550 ppb), these polymers rapidly form a filterable precipitate upon metal capture, reducing the concentration of cation to <1.5 ppb within 3 min, as measured by inductively coupled plasma mass spectrometry. This system effectively removes cadmium ions from highly contaminated solutions to levels below the Agency for Toxic Substances and Disease Registry limit for Cd2+ in drinking water and selectively removes both Cd2+ and Pb2+ from lake water spiked with trace amounts of metal. Acidification triggers protonation of the glucuronate groups, releasing the heavy metals and resolubilizing the polymer. This capture-and-release process can be repeated over multiple cycles without loss of binding capacity. As such, this study introduces a novel class of recyclable materials with pH-responsive properties, offering potential for applications in water remediation and beyond.

Durability of Mortars Manufactured with Low-Carbon Binders Exposed to Calcium Chloride-Based De-Icing Salts

Calcium chloride is one of the main de-icing salts for removing snow and ice from roads, infrastructures and service areas. It is well known that reinforced concrete structures, if exposed to calcium chloride, can suffer from severe damages due to both corrosion of steel reinforcement and chemical attack of the cement paste. This paper aims at evaluating the resistance to chemical attack of mortars manufactured with different low-carbon binders (alkali activated slag cements, calcium sulphoaluminate cement-based blends, high volume ultrafine fly ashes cements) in presence of CaCl2-based de-icing salts in cold weather (temperature about 4°C). Results indicated that alkali activated slag-based mortars are quasi-immune to calcium chloride attack due to their mineralogical composition. On the contrary, calcium sulphoaluminate-based blends show the total loss of binding capacity, especially when calcium sulphoaluminate cement is used with gypsum and Portland cement. Finally, the partial substitution of Portland cement with ultrafine fly ash strongly reduces the mass change and the strength loss of mortars submerged in 30 wt.% CaCl2 solutions due to the strong reduction of calcium hydroxide responsible for the calcium oxychloride formation in the cement paste.

Insight into purification of monoclonal antibodies in industrial columns via studies of Protein A binding capacity by in situ ATR-FTIR spectroscopy.

Therapeutic monoclonal antibodies (mAbs) are effective treatments for a range of cancers and other serious diseases, however mAb treatments cost on average ∼$100 000 per year per patient, limiting their use. Currently, industry favours Protein A affinity chromatography (PrAc) as the key step in downstream processing of mAbs. This step, although highly efficient, represents a significant mAb production cost. Fouling of the Protein A column and Protein A ligand leaching contribute to the cost of mAb production by shortening the life span of the resin. In this study, we assessed the performance of used PrAc resin recovered from the middle inlet, center and outlet as well as the side inlet of a pilot-scale industrial column. We used a combination of static binding capacity (SBC) analysis and Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopy to explore the used resin samples. SBC analysis demonstrated that resin from the inlet of the column had lower binding capacity than resin from the column outlet. ATR-FTIR spectroscopy with PLS (partial least square) analysis confirmed the results obtained from SBC analysis. Importantly, in situ ATR-FTIR spectroscopy also allowed both measurement of the concentration and assessment of the conformational state of the bound Protein A. Our results reveal that PrAc resin degradation after use is dependent on column location and that neither Protein A ligand leaching nor denaturation are responsible for binding capacity loss.

Affinity purification of human alpha galactosidase utilizing a novel small molecule biomimetic of alpha-D-galactose

Alpha galactosidase (a-Gal) is an acidic hydrolase that plays a critical role in hydrolyzing the terminal alpha-galactoyl moiety from glycolipids and glycoproteins. There are over a hundred mutations reported for the GLA gene that encodes a-Gal that result in reduced protein synthesis, protein instability, and reduction in function. The deficiencies of a-Gal can cause Fabry disease, a rare lysosomal storage disorder (LSD) caused by the failure to catabolize alpha-d-galactoyl glycolipid moieties. The current standard of care for Fabry disease is enzyme replacement therapy (ERT) where the purified recombinant form of human a-Gal is given to patients. The manufacture of a-Gal is currently performed utilizing traditional large-scale chromatography processes. Developing an affinity resin for the purification of a-Gal would reduce the complexity of the manufacturing process, reduce costs, and potentially produce a higher quality a-Gal. After the evaluation of many small molecules, a commercially available small molecule biomimetic, N-5-Carboxypentyl-1-deoxygalactonojirimycin (N5C-DGJ), was utilized for the development of a novel small molecule biomimetic affinity resin for a-Gal. Affinity purified a-Gal demonstrated a purity greater than 90%, exhibited expected thermal stability and specific activity. Complementing this affinity purification is the development of an elution buffer system that confers an increased thermal stability to a-Gal. The N5C-DGJ affinity resin tolerated sodium hydroxide sanitization with no loss of binding capacity, making it amenable to large scale purification processes and potential use in manufacturing. This novel method for purifying the challenging a-Gal enzyme can be extended to other enzyme replacement therapies.

Heterozygous LHX3 mutations may lead to a mild phenotype of combined pituitary hormone deficiency.

LHX3 is an LIM domain transcription factor involved in the early steps of pituitary ontogenesis. We report here functional studies of three allelic variants, including the first heterozygous variant of LHX3 NM_178138.5(LHX3):c.587T>C (p.(Leu196Pro)) that may be responsible for a milder phenotype of hypopituitarism. Our functional studies showed that NM_178138.5(LHX3):c.587T>C (p.(Leu196Pro)) was not able to activate target promoters in vitro, as it did not bind DNA, and likely affected LHX3 function via a mechanism of haplo-insufficiency. Our study demonstrates the possibility that patients with a heterozygous variant of LHX3 may have pituitary deficiencies, with a milder phenotype than patients with homozygous variants. It is thus of vital to propose an optimal follow-up of such patients, who, until now, were considered as not being at risk of presenting pituitary deficiency. The second variant NM_178138.5(LHX3):c.622C>G (p.(Arg208Gly)), present in a homozygous state, displayed decreased transactivating ability without loss of binding capacity in vitro, concordant with in silico analysis; it should thus be considered to affect LHX3 function. In contrast, the NM_178138.5(LHX3):c.929G>C (p.(Arg310Pro)) variant, in a heterozygous state, also predicted as deleterious in silico, proved functionally active in vitro, and should thus still be classified as a variant of unknown significance. Our study emphasizes the need for functional studies due to the limits of software-based predictions of new variants, and the possible association of a pituitary phenotype to heterozygous LHX3 variants.

Ultrafast and high-capacity adsorption of Gd(III) onto inorganic phosphorous acid modified mesoporous SBA-15

The development of rare earth adsorbent with high adsorption capacity and rapid adsorption rate is one of the most important issues for enriching and recovering rare earth ions. In the present work, an inorganic phosphorous acid-modified mesoporous SBA-15 (P-SBA-15) was facilely synthesized by a simple and cost-effective post-grafting approach, and its adsorption behavior towards the rare earth ion Gd(III) was investigated. Benefiting from high specific surface area (669.7m2·g−1), large pore size (9.1nm), and the presence of abundant phosphorous acid groups (1.4mmol·g−1) on the surface, the P-SBA-15 exhibited an excellent performance in terms of capacity and kinetics on adsorption of Gd(III). Under optimized conditions, the adsorption capacity of P-SBA-15 towards Gd(III) was up to 1.3mmol·g−1 at 30°C, which is the second highest value as compared with previously reported Gd(III) adsorbents. Moreover, the adsorption of Gd(III) onto P-SBA-15 was ultrafast, achieving adsorption equilibrium within only 2min. Test of reusability revealed that this mesoporous adsorbent could be repeatedly used several times without significant loss in binding capacity. This work not only provides a new insight into the fabrication of phosphorous acid-functionalized mesoporous silica, but also demonstrates its prospective application in adsorptive removal and/or recovery of rare earth ions.

Hierarchical porous molecule/ion imprinted polymers with double specific binding sites: Combination of Pickering HIPEs template and pore-filled strategy

To recognize and remove targeted organic and inorganic pollutants simultaneously, a simple and outstanding approach was provided to fabricate hierarchical porous molecule/ion imprinted polymers (M-IIPs) with double specific binding sites, Firstly, λ-cyhalothrin (LC) molecules imprinted polymer foam initiators (MIPFIs) with the average void diameter of 8.0μm were prepared via a W/O Pickering high internal phase emulsions (HIPEs) template stabilized by vinyl-silica particles (V-SPs). Secondly, based on a pore-filled strategy, adjustable amounts of copper ions (Cu(II)) imprinted polymers (IIPs) were incorporated into the macroporous surface of MIPFIs via atom transfer free radical polymerization (ATRP). Owing to the well-defined hierarchical porous structure, M-IIPs possessed enhanced binding kinetics, and the maximum monolayer adsorption capacities of as-prepared M-IIPs were 193.1μmolg−1 and 169.9μmolg−1 towards LC and Cu(II), respectively. The imprinting factor ε was 1.942 for LC, while their relative selectivity coefficients αr for Cu(II)/Zn(II) and Cu(II)/Ni(II) were 6.31 and 4.24 times higher than those of non-imprinted polymers (N-NIPs), illustrating that M-IIPs showed excellent molecular recognition in testing solutions. In the binary solution of LC and Cu(II), an enhanced adsorption capacities towards LC and Cu(II) were observed, indicating the existence of promotion in selective adsorption ability in the case of coexistence of LC and Cu(II). Besides, test of reusability suggested that the M-IIPs could be repeatedly used without any significant loss in binding capacity. Overall, this work not only provided new insights into the fabrication of porous imprinted composites with huge specific surface area and binary function, but also highlighted their application for selective and simultaneous removal of LC and Cu(II).

Nanostructured Membranes from Triblock Polymer Precursors as High Capacity Copper Adsorbents.

Membrane adsorbers are a proposed alternative to packed beds for chromatographic separations. To date, membrane adsorbers have suffered from low binding capacities and/or complex processing methodologies. In this work, a polyisoprene-b-polystyrene-b-poly(N,N-dimethylacrylamide) (PI-PS-PDMA) triblock polymer is cast into an asymmetric membrane that possesses a high density of nanopores (d ∼ 38 nm) at the upper surface of the membrane. Exposing the membrane to a 6 M aqueous hydrochloric acid solution converts the PDMA brushes that line the pore walls to poly(acrylic acid) (PAA) brushes, which are capable of binding metal ions (e.g., copper ions). Using mass transport tests and static binding experiments, the saturation capacity of the PI-PS-PAA membrane was determined to be 4.1 ± 0.3 mmol Cu(2+) g(-1). This experimental value is consistent with the theoretical binding capacity of the membranes, which is based on the initial PDMA content of the triblock polymer precursor and assumes a 1:1 stoichiometry for the binding interaction. The uniformly sized nanoscale pores provide a short diffusion length to the binding sites, resulting in a sharp breakthrough curve. Furthermore, the membrane is selective for copper ions over nickel ions, which permeate through the membrane over 10 times more rapidly than copper during the loading stage. This selectivity is present despite the fact that the sizes of these two ions are nearly identical and speaks to the chemical selectivity of the triblock polymer-based membrane. Furthermore, addition of a pH 1 solution releases the bound copper rapidly, allowing the membrane to be regenerated and reused with a negligible loss in binding capacity. Because of the high binding capacities, facile processing method implemented, and ability to tailor further the polymer brushes lining the pore walls using straightforward coupling reactions, these membrane adsorbers based on block polymer precursors have potential as a separation media that can be designed to a variety of specific applications.

Selective Adsorption of Gd(3+) on a Magnetically Retrievable Imprinted Chitosan/Carbon Nanotube Composite with High Capacity.

A novel magnetic imprinting nanotechnology for selective capture of Gd(3+) from a mixed solution of rare earth ions was developed by simply adding Gd(3+)-imprinted chitosan/carbon nanotube nanocomposite (IIP-CS/CNT) and silica-coated magnetite nanoparticle (SiO2@Fe3O4). The IIP-CS/CNT was prepared for the first time via a facile "surface deposition-crosslinking" method, exhibiting a well-defined coating structure. Interestingly, the neighboring IIP-CS/CNT monomers were held together as bundles, like a network, containing abundant interstitial spaces. When IIP-CS/CNT and SiO2@Fe3O4 were dispersed in a mixed solution of rare earth ions, the magnetic SiO2@Fe3O4 submicrospheres would be trapped in or adhere to the IIP-CS/CNT network, leading to the magnetization of IIP-CS/CNT; meanwhile, Gd(3+) ions could be selectively captured by the magnetized IIP-CS/CNT. Saturation adsorption capacity for Gd(3+) was up to 88 mg g(-1) at 303.15 K, which is significantly higher than the Gd(3+) adsorption capacities for the reported rare earth ion-imprinted adsorbents over recent years. The selectivity coefficients relative to La(3+) and Ce(3+) were 3.50 and 2.23, respectively, which are very similar to those found for other reported CS-based imprinted materials. Moreover, the imprinted adsorbents could be easily and rapidly retrieved by an external magnetic field without the need of additional centrifugation or filtration, greatly facilitating the separation process. Test of reusability demonstrated that the magnetized IIP-CS/CNT could be repeatedly used without any significant loss in binding capacity. Overall, this work not only provides new insights into the fabrication of magnetic imprinted CS-based composite, but also highlights its application for selective adsorption toward rare earth ions.

Development of transient phage resistance in Campylobacter coli against the group II phage CP84.

Recently, there is a growing interest in the use of bacteriophages for pre- and post-harvest applications to reduce foodborne pathogens (including Campylobacter) along the food chain. Quantitative Campylobacter reductions of up to three log10 units have been achieved by phage application. However, possible phage resistance might limit this approach. In Campylobacter (C.) jejuni, phage resistance mechanisms have been described in detail but data on these mechanisms in C. coli are still missing. To study phage resistance in C. coli, strain NCTC 12668 was infected with the lytic phage CP84, belonging to group II of Campylobacter phages. Resistant and sensitive clones were analysed using phenotypic and genotypic assays. C. coli clones acquired only transient resistance against CP84. The resistance led to cross-protection to one out of five other group II phages tested. Phage resistance was apparently neither caused by large genomic rearrangements nor by a CRISPR system. Binding assays demonstrated that CP84 could not adsorb to resistant C. coli clones suggesting a bacterial phage receptor to be involved in resistance. However, phage resistant C. coli clones did not reveal an altered motility or modified flaA sequence. Considering the loss of binding capacity and the reversion to a phage sensitive phenotype we hypothesize that acquired resistance depends on temporal phase variable switch-off modifications of the phage receptor genes, even though the resistance mechanism could not be elucidated in detail. We further speculate that even closely related phages of the same group use different bacterial receptors for binding on C. coli.

Evaluation of a novel methacrylate-based Protein A resin for the purification of immunoglobulins and Fc-fusion proteins.

Protein A affinity chromatography is a central part of most commercial monoclonal antibody and Fc-fusion protein purification processes. In the last couple years an increasing number of new Protein A technologies have emerged. One of these new Protein A technologies consists of a novel, alkaline-tolerant, Protein A ligand coupled to a macroporous polymethacrylate base matrix that has been optimized for immunoglobulin (Ig) G capture. The resin is interesting from a technology perspective because the particle size and pore distribution of the base beads are reported to have been optimized for high IgG binding and fast mass transfer, while the Protein A ligand has been engineered for enhanced alkaline tolerance. This resin was subjected to a number of technical studies including evaluating dynamic and static binding capacities, alkaline stability, Protein A leachate propensity, impurity clearance, and pressure–flow behavior. The results demonstrated similar static binding capacities as those achieved with industry standard agarose Protein A resins, but marginally lower dynamic binding capacities. Removal of impurities from the process stream, particularly host cell proteins, was molecule dependent, but in most instances matched the performance of the agarose resins. This resin was stable in 0.1 M NaOH for at least 100 h with little loss in binding capacity, with Protein A ligand leakage levels comparable to values for the agarose resins. Pressure–flow experiments in lab-scale chromatography columns demonstrated minimal resin compression at typical manufacturing flow rates. Prediction of resin compression in manufacturing scale columns did not suggest any pressure limitations upon scale up. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1125–1136, 2014

Preparation of Imprinted PVB/β‐CD Nanofiber by Electrospinning Technique and Its Selective Binding Abilities for Naringin

AbstractA novel molecularly imprinted composite nanofiber was prepared by a simple electrospinning technique, in which polyvinylbutyral (PVB) was chosen as matrix, β‐cyclodextrin (β‐CD) was used as a functional monomer and naringin (NG) as template molecules. After cross‐linked by hexamethylene diisocyanate (HMDI), the composite nanofiber exhibited a high specific binding capacity. The morphological structure of the nanofibers was studied by means of infrared spectrum (IR), X‐ray diffraction (XRD), and scanning electron microscopy (SEM). The β‐CD molecules were mostly homogeneously distributed within the PVB nanofiber without forming phase separated crystalline aggregates. Compared with traditional imprinted β‐CD polymer, the binding experiments demonstrated that the molecularly imprinted composite nanofiber shows the specific binding sites and the selective binding ability for NG. The molecularly imprinted nanofiber could be used at least six times without any loss in binding capacity.

Biphenyl sorption to different soil clay minerals

Only limited information is available in the literature on the role of soil mineral surfaces in the sorption of hydrophobic organic pollutants. This knowledge gap is addressed through the assessment of biphenyl sorption to kaolinite, illite and bentonite; using the batch equilibration technique with incubations lasting 6 or 21 days at 28 ± 2°C in the dark [RT1]. Sorption of biphenyl onto kaolinite followed the Freundlich sorption isotherm, whereas linear sorption isotherms were observed on illite and bentonite [RT2]. The biphenyl sorption partition coefficient on kaolinite ranged from 0.1 to 9.1 cm3.g-1 after 6 days and no sorption was observed after 21 days. This could have been caused by a completely reversible sorption or a loss of binding capacity after 21 days [RT3]. The respective values of the biphenyl sorption partition coefficient on illite and bentonite ranged from 20.3 ± 0.3 to 120 ± 8 cm3.g-1.Sorption equilibrium on the internal clay surfaces was reached after 6 days, as indicated by the sorption data for illite and bentonite [RT4]. Access of biphenyl molecules to the internal clay surfaces is a function of the ionic strength of soil solution and the soil organic matter is the dominant site for biphenyl sorption after 6 to 21 days. Key words: Biphenyl, sorption, kaolinite, illite, bentonite.

Solid‐phase preparation of protein complexes

Protein-protein conjugation is usually achieved by solution phase methods requiring concentrated protein solution and post-synthetic purification steps. In this report we describe a novel continuous-flow solid-phase approach enabling the assembly of protein complexes minimizing the amount of material needed and allowing the repeated use of the same solid phase. The method exploits an immunoaffinity matrix as solid support; the matrix reversibly binds the first of the complex components while the other components are sequentially introduced, thus allowing the complex to grow while immobilized. The tethering technique employed relies on the use of the very mild synthetic conditions and fast association rates allowed by the avidin-biotin system. At the end of the assembly, the immobilized complexes can be removed from the solid support and recovered by lowering the pH of the medium. Under the conditions used for the sequential complexation and recovery, the solid phase was not damaged or irreversibly modified and could be reused without loss of binding capacity. The method was specifically designed to prepare protein complexes to be used in immunometric methods of analysis, where the immunoreactivity of each component needs to be preserved. The approach was successfully exploited for the preparation of two different immunoaffinity reagents with immunoreactivity mimicking native squamous cell carcinoma antigen-immunoglobulin M (SCCA-IgM) and alphafetoprotein-immunoglobulin M (AFP-IgM) immune complexes, which were characterized by dedicated sandwich enzyme-linked immunosorbent assay (ELISA) and immunoblot. Besides the specific application described in the paper, the method is sufficiently general to be used for the preparation of a broad range of protein assemblies.

Heterodimerization of the Sialidase NEU1 with the Chaperone Protective Protein/Cathepsin A Prevents Its Premature Oligomerization

Lysosomal neuraminidase-1 (NEU1) forms a multienzyme complex with beta-galactosidase and protective protein/cathepsin A (PPCA). Because of its association with PPCA, which acts as a molecular chaperone, NEU1 is transported to the lysosomal compartment, catalytically activated, and stabilized. However, the mode(s) of association between these two proteins both en route to the lysosome and in the multienzyme complex has remained elusive. Here, we have analyzed the hydrodynamic properties of PPCA, NEU1, and a complex of the two proteins and identified multiple binding sites on both proteins. One of these sites on NEU1 that is involved in binding to PPCA can also bind to other NEU1 molecules, albeit with lower affinity. Therefore, in the absence of PPCA, as in the lysosomal storage disease galactosialidosis, NEU1 self-associates into chain-like oligomers. Binding of PPCA can reverse self-association of NEU1 by causing the disassembly of NEU1-oligomers and the formation of a PPCA-NEU1 heterodimeric complex. The identification of binding sites between the two proteins allowed us to create innovative structural models of the NEU1 oligomer and the PPCA-NEU1 heterodimeric complex. The proposed mechanism of interaction between NEU1 and its accessory protein PPCA provides a rationale for the secondary deficiency of NEU1 in galactosialidosis.

Structural and Functional Study of Mouse Antidigoxin Monoclonal Antibody Against Thermal Variation

Conformation, heat-induced conformational changes, and the stability of mouse monoclonal antibody against digoxin were analyzed by circular dichroism (CD), fluorescence, UV-Vis spectroscopy, and ELISA techniques. Since both biological function and aggregation depend on the conformational properties of mouse IgG molecules, a series of sensitive and rapid physical methods were introduced to assess conformational changes and structural stability as a function of temperature. CD spectra of IgG show the characteristics expected for beta-proteins. Denaturation experiments performed through optical spectroscopic methods and affinity measurement indicated that the antibody unfolds cooperatively in a single transition. Thus, unfolding/refolding equilibrium proceeds via a simple two-state mechanism (N equilibrium U), where only the native and the unfolded states are significantly populated. Thermal-induced denaturation, however, is not completely reversible, and the partial loss of binding capacity might be due, at least in part, to incorrect refolding of the long loops (CDRs), which are responsible for antigen recognition. The results show that purified IgG is temperature stable. Of course, affinity assessment as measured by ELISA indicated that increasing the temperature decreases the affinity of IgG toward digoxin.

Synthesis and Application of a Novel Ligand for Affinity Chromatography Based Removal of Endotoxin from Antibodies

Endotoxin or lipopolysaccharide (LPS) contamination in proteins expressed by Gram-negative bacteria is a major drawback associated with protein expression. Endotoxin intoxication in humans and animals above a certain threshold level can result in a fatal immune response. Reduction in endotoxin levels is therefore essential before proteins can be used in in vivo studies or sold as pharmaceutical products. Affinity chromatography employing the peptide Polymyxin B (PMB) as an affinity ligand is one way in which endotoxin contamination has been addressed; this is, however, a costly process. We describe the synthesis of a novel affinity ligand based on the structure of the drug pentamidine, which can be applied effectively in endotoxin removal. The synthetic route to this ligand is straightforward and inexpensive, while the ligand can be readily immobilized onto activated sepharose beads. Thus, we demonstrate that these pentamidine affinity beads bind endotoxin/LPS with comparable capacity to PMB affinity systems, that the beads can be recycled efficiently and economically without loss of binding capacity, and application of the functionalized beads for endotoxin removal in an authentic contaminated antibody sample.

A molecular imprinted membrane for molecular discrimination of tetracycline hydrochloride

Membranes of poly(acrylonitrile-co-acrylic acid) made by phase inversion technique in the presence of tetracycline hydrochloride showed molecular recognition behavior towards the template molecule. The interactions of the carboxylic groups of the polymer chain played an important role in the discrimination process. Prolonged washing with distilled water regenerated the membrane without any loss of binding capacity. Chloramphenicol, of similar polarity and fair solubility in water, is recognized less efficiently.

Direct detection of the binding of avidin and lactoferrin fluorescent probes to heparinized surfaces

We describe the use of two heparin-binding proteins, avidin and lactoferrin, as probes for monitoring the amount of heparin immobilized to plastic surfaces. The proteins were derivatized with either fluorescent labels or europium chelates, enabling sensitive, fast, reproducible, and robust assays, and were used to measure the amount of protein bound to heparinized microplates, with particular attention to plates that have been coated with bovine serum albumin (BSA)–heparin conjugate. This direct method unequivocally shows that BSA–heparin affords an economical, convenient, and reliable method for coating both polystyrene microtiter plates and magnetic beads with heparin. We demonstrate that assays using directly labeled proteins overcome the problems of dissociation of the heparin–protein complex, which can occur during incubation and washing steps associated with antibody-based detection methods, and the loss in binding capacity caused by certain blocking regimes. We suggest that labeled avidin and lactoferrin are convenient probes for heparinized surfaces with the potential for much wider applicability than that presented here.

Site-specific and reversible anchoring of active proteins onto cellulose using a cellulosome-like complex

Protein engineering strategies facilitating controlled and spontaneous assembly of macromolecular complexes are of great interest for the design of artificial multi-enzyme systems of pre-defined composition. Here we have combined affinity proteins from different sources to achieve specific and reversible anchoring of affinity domain-tagged reporter proteins to a cellulose-anchored fusion protein. The design principle mimics the architecture of macromolecular cellulosome complexes produced by some cellulolytic microbes. A fusion protein between a cellulose-binding module (CBM1Cel6A) of the Trichoderma reesei cellobiohydrolase Cel6A and a five-domain staphylococcal protein A (SPA) was constructed to serve as platform for docking of easily detectable reporter proteins onto cellulose surfaces. In turn, the reporter proteins were produced as fusions to two copies of a SPA-binding affinity protein (an affibody denoted ZSPA-1), selected from a phage display library constructed by combinatorial protein engineering. In a series of experiments, involving repeated washing and low pH elution, affinity-tagged Enhanced Green Fluorescent Protein (EGFP) and Fusarium solani pisi lipase cutinase reporter proteins were both found to be specifically directed from solution to the same region of a cellulose filter paper where SPA-CBM1Cel6A fusion protein had been previously applied. This showed that the SPA-CBM1Cel6A fusion protein had been stably anchored to the cellulose surface without loss of binding capacity and that the interaction between SPA and the ZSPA-1 affibody domains was selective. The generality of this biospecificity-driven system for assembly applications is discussed.