Arginine Inhibits Adsorption of Proteins on Polystyrene Surface

Nonspecific adsorption of protein on solid surfaces causes a reduction of concentration as well as enzyme inactivation during purification and storage. However, there are no versatile inhibitors of the adsorption between proteins and solid surfaces at low concentrations. Therefore, we examined additives for the prevention of protein adsorption on polystyrene particles (PS particles) as a commonly-used material for vessels such as disposable test tubes and microtubes. A protein solution was mixed with PS particles, and then adsorption of protein was monitored by the concentration and activity of protein in the supernatant after centrifugation. Five different proteins bound to PS particles through electrostatic, hydrophobic, and aromatic interactions, causing a decrease in protein concentration and loss of enzyme activity in the supernatant. Among the additives, including arginine hydrochloride (Arg), lysine hydrochloride, guanidine hydrochloride, NaCl, glycine, and glucose, Arg was most effective in preventing the binding of proteins to PS particles as well as activity loss. Moreover, even after the mixing of protein and PS particles, the addition of Arg caused desorption of the bound protein from PS particles. This study demonstrated a new function of Arg, which expands the potential for application of Arg to proteins.     

Mechanism of protein extraction from the solid state by water-in-oil microemulsions

The extraction of solid-phase alpha-chymotrypsin, bovine serum albumin (BSA), and lysozyme by water-in-oil microemulsion (w/o-ME) solution containing Aerosol-OT (AOT) was thoroughly examined as a means to maximize protein solubilization in organic solvent media. Protein extraction occurred simultaneously with the adsorption of water and AOT by the solid protein. Water and AOT were desorbed at nearly equal rates, suggesting that both materials were desorbed together as micreomulsions. The solubilization of protein increased linearly with the ratio of solid protein to extractant solution except at a high value of the ratio, where most protein-containing microemulsions were desorbed. Based on our results, a mechanistic model was developed to describe the solid-phase extraction procedure. First, microemulsions are desorbed from solution by the solid protein, resulting in the formation of a solid protein-AOT-water aggregate. Second, when a protein in the solid phase binds to a sufficient number of microemulsions, the resulting aggregate's increased hydrophobicity drives its solubilization into lipophilic solvent. Third, through the exchange of materials between the solubilized precipitate and the remaining microemulsions, protein-containing w/o-MEs are formed. The presence of adsorption is further indicated by an isotherm existing between the water, AOT, and protein content of the resulting solid phase for each protein. The driving force behind adsorption is either AOT-protein interactions or the protein's affinity for microemulsion-encapsulated water, depending on the properties of the protein and the size of the microemulsions, in agreement with the model of P. L. Luisi [Chimia, 44: 270-282 (1990)]. The second step of our model is mass transfer limited for the extraction of solid alpha-chymotrypsin and BSA. The extraction of solid lysozyme was limited by the occurrence of an irreversible precipitation process. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 583-593, 1997.     

Characterization of the diatomite binding domain in the ribosomal protein L2 from E. coli and functions as an affinity tag

The ribosomal protein L2, a constituent protein of the 50S large ribosomal subunit, can be used as Si-tag using silica particles for the immobilization and purification of recombinant proteins (Ikeda et al. (Protein Expr Purif 71:91–95, 2010); Taniguchi et al. (Biotechnol Bioeng 96:1023–1029, 2007)). We applied a diatomite powder, a sedimentary rock mainly composed with diatoms silica, as an affinity solid phase and small ubiquitin-like modifier (SUMO) technology to release a target protein from the solid phase. The L2 (203–273) was the sufficient region for the adsorption of ribosomal protein L2 on diatomite. We comparatively analyzed the different adsorption properties of the two deleted proteins of L2 (L2 (1–60, 203–273) and L2 (203–273)) on diatomite. The time required to reach adsorption equilibrium of L2 (203–273) fusion protein on diatomite was shorter than that of L2 (1–60, 203–273) fusion protein. The maximum adsorption capacity of L2 (203–273) fusion protein was larger than that of L2 (1–60, 203–273) fusion protein. In order to study whether the L2 (203–273) can function as an affinity purification tag, SUMO was introduced as one specific protease cleavage site between the target protein and the purification tags. The L2 (203–273) and SUMO fusion protein purification method was tested using enhanced green fluorescent protein as a model protein; the result shows that the purification performance of this affinity purification method was good. The strong adsorption characteristic of L2 (203–273) on diatomite also provides a potential protein fusion tag for the immobilization of enzyme.     

Solid phase extraction of the zwitterionic detergent chaps

Multiple techniques for solid phase adsorption of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) were evaluated. Both the porous polystyrene divinylbenzene matrices (BioBeads SMTM) and Extracti GelTM D reduced CHAPS to significantly below its critical micellar concentration while Extracti-GelTM removed CHAPS to below detectable limits. Bio-Bead extraction of CHAPS correlated with the surface area of the bead type. SM-16 beads, with the largest effective surface area, removed nearly 97% of the detergent. For a given amount of detergent and mass of Bio-Beads, the ratio of sample to total bead volume significantly affected CHAPS adsorption. Total protein recovery with the Extracti-GelTM was approximately 97%. Protein recovery in the samples treated with Bio-Beads varied from 56-95%. Chromatographic rather than batch processing yielded optimum recoveries. CHAPS can be effectively removed from dilute protein solutions by solid phase adsorption and this technique offers significant advantages over standard dialysis or gel filtration methods.     

Measuring adsorption of a hydrophobic probe with a surface plasmon resonance sensor to monitor conformational changes in immobilized proteins

Conformational changes of proteins immobilized on solid matrices were observed by measuring the adsorption of Triton X-100 (TX), a nonionic detergent, as a hydrophobic probe with BIACORE, a biosensor that utilizes the phenomenon of surface plasmon resonance (SPR). Two kinds of proteins, alpha-glucosidase and lysozyme, were covalently attached to dextran matrices on the sensor surface in the flow cell and then exposed to various concentrations of TX solution. We measured SPR signal changes derived from adsorption of TX to the immobilized proteins and calculated the monolayer adsorption capacity using the Brunauer-Emmett-Teller (BET) equation. The results demonstrated that monolayer adsorption capacity is proportional to the amount of immobilized proteins. Further, the unfolding process of immobilized proteins on the sensor surface induced by guanidine hydrochloride was investigated by monitoring SPR signal increases due to the adsorption of TX to the exposed hydrophobic region of the protein. Results strongly suggested that the increase in the SPR signal reflected the formation of the agglutinative unfolded state. We expect our measuring method using the SPR sensor and TX adsorption will be a novel tool to provide conformational information regarding various proteins on solid matrices.     

Protein adsorption at solid-liquid interfaces: Part IV--Effects of different solid-liquid systems and various neutral salts.

Adsorption isotherms of BSA at the solid-water interfaces have been studied as a function of protein concentration, ionic strength of the medium, pH and temperature using silica, barium sulphate, carbon, alumina, chromium, ion-exchange resins and sephadex as solid interfaces. In most cases, isotherms for adsorption of BSA attained the state of adsorption saturation. In the presence of barium sulphate, carbon and alumina, two types in the isotherms are observed. Adsorption of BSA is affected by change in pH, ionic strength and temperature of the medium. In the presence of metallic chromium, adsorbed BSA molecules are either denatured or negatively adsorbed at the metallic interface. Due to the presence of pores in ion-exchange resins, adsorption of BSA is followed by preferential hydration on resin surfaces in some cases. Sometimes two steps of isotherms are also observed during adsorption of BSA on the solid resins in chloride form. Adsorption of BSA, beta-lactoglobulin, gelatin, myosin and lysozyme is negative on Sephadex surface due to the excess adsorption of water by Sephadex. The negative adsorption is significantly affected in the presence of CaCl2, KSCN, LiCl, Na2SO4, NaI, KCl and urea. The values of absolute amounts of water and protein, simultaneously adsorbed on the surface of different solids, have been evaluated in some cases on critical thermodynamic analysis. The standard free energies (delta G0) of excess positive and negative adsorption of the protein per square meter at the state of monolayer saturation have been calculated using proposed universal scale of thermodynamics. The free energy of adsorption with reference to this state is shown to be strictly comparable to each other. The magnitude of standard free energy of transfer (delta G0B) of one mole of protein or a protein mixture at any type of physiochemical condition and at any type of surface is observed to be 38.5 kJ/mole.     

Protein adsorption orientation in the light of fluorescent probes: mapping of the interaction between site-directly labeled human carbonic anhydrase II and silica nanoparticles

Little is known about the direction and specificity of protein adsorption to solid surfaces, a knowledge that is of great importance in many biotechnological applications. To resolve the direction in which a protein with known structure and surface potentials binds to negatively charged silica nanoparticles, fluorescent probes were attached to different areas on the surface of the protein human carbonic anhydrase II. By this approach it was clearly demonstrated that the adsorption of the native protein is specific to limited regions at the surface of the N-terminal domain of the protein. Furthermore, the adsorption direction is strongly pH-dependent. At pH 6.3, a histidine-rich area around position 10 is the dominating adsorption region. At higher pH values, when the histidines in this area are deprotonated, the protein is also adsorbed by a region close to position 37, which contains several lysines and arginines. Clearly the adsorption is directed by positively charged areas on the protein surface toward the negatively charged silica surface at conditions when specific binding occurs.     

Application of vortex flow adsorption technology to intein-mediated recovery of recombinant human alpha1-antitrypsin

Vortex flow is a secondary flow pattern that appears above a critical rotation rate in the annular gap between an inner rotating solid cylinder and an outer stationary cylindrical shell. By suspending adsorbent resin in the vortices, a novel unit operation, vortex flow adsorption (VFA), is created. In VFA, the rotation of the inner cylinder facilitates the fluidization of the adsorbent resin. Similar to expanded bed processes, VFA has high fluid voidage so that it can be used to recover biochemical products directly from fermentation broths or cell homogenates without removing cells or cell debris first. In this study, recombinant human alpha1-antitrypsin (alpha1-AT) was expressed in Escherichia coli as a fusion with a modified intein containing a chitin-binding domain. Therefore, the fusion protein can be recovered by chitin resin affinity adsorption. The intein can be induced to undergo in vitro peptide bond cleavage to specifically release alpha1-AT from the bound fusion protein. The capture efficiency of the fusion protein, 26.2%, was obtained in the VFA process. In addition, the specific activity of alpha1-AT was dramatically improved from 0.3 to 205.2 EIC/(mg total protein) after adsorption and cleavage. Therefore, vortex flow adsorption is an integrative technology to combine the primary clarification, concentration, and purification steps in conventional downstream processing into a single unit operation to efficiently recover and purify biochemical products.     

Spatially controlled electro-stimulated DNA adsorption and desorption for biochip applications

The manipulation of biomolecules at solid/liquid interfaces is important for the enhanced performance of a number of biomedical devices, including biochips. This study focuses on the spatial control of surface interactions of DNA as well as the electro-stimulated adsorption and desorption of DNA by appropriate surface modification of highly doped p-type silicon. Surface modification by plasma polymerisation of allylamine resulted in a surface that supported DNA adsorption and sustained cell attachment. Subsequent high-density grafting of poly(ethylene oxide) formed a low fouling layer resistant to biomolecule adsorption and cell attachment. Spatially controlled excimer laser ablation of the surface produced patterns of re-exposed plasma polymer with high-resolution. On patterned surfaces, preferential electro-stimulated adsorption of DNA to the allylamine plasma polymer surface and subsequent desorption by the application of a negative bias was observed. Furthermore, the concept presented here was investigated for use in transfection chips. Cell culture experiments with human embryonic kidney cells, using the expression of green fluorescent protein as a reporter, demonstrated efficient and controlled transfection of cells. Electro-stimulated desorption of DNA was shown to yield significantly enhanced solid phase transfection efficiencies to values of up to 30%. The ability to spatially control DNA adsorption combined with the ability to control the binding and release of DNA by application of a controlled voltage enables an advanced level of control over DNA bioactivity on solid substrates and lends itself to biochip applications.     

Protein interactions with surfaces: Computational approaches and repellency

Study of protein adsorption to solid surfaces continues to be substantial because of its role in cellular responses to biomaterials, interest in molecular aspects such as conformation and orientation, new methods for making protein repellent surfaces, and new application areas such as nanoparticles and microfluidics. This brief review is based only on very recent articles of particular interest to the authors, who each have worked in this area for some time. Simulations of protein interactions with surfaces and protein repellent surfaces are the only subtopics reviewed here.     

Interaction of calcium ions and salivary acidic proline-rich proteins with hydroxyapatite. A possible aspect of inhibition of hydroxyapatite formation.

The relationship between Ca2+- and hydroxyapatite-binding sites in salivary acidic proline-rich phosphoproteins A and C was investigated. Coating of hydroxyapatite with protein before adsorption had no effect on Ca2+ binding to the mineral, but simultaneous adsorption of Ca+ and protein to hydroxyapatite caused additional Ca2+ binding to the solid. The additional amount of Ca2+ adsorbed, measured in mol of Ca2+/mol of protein adsorbed to hydroxyapatite, was approx. 2 for protein C, 4 for protein A, 9 for the N-terminal tryptic peptide and 2 for dephosphorylated protein A. It is suggested that the ability of the proteins to inhibit hydroxyapatite formation is related to the binding of the proteins to crystal growth sites on the mineral, which prevents access of Ca2+ from the surrounding liquid.     

Dynamic behavior of adsorber membranes for protein recovery

In recent years there has been a considerable interest in developing membrane chromatography systems that function as a short, wide chromatographic column in which the adsorptive packing consists of one or more microporous membranes. This study reports the use of new adsorber membranes prepared by the incorporation of various types of ion exchange resins into an EVAL porous membrane for protein recovery. The obtained heterogeneous matrixes composed of solid particles surrounded by the polymeric film possess a good accessibility for the protein to the adsorptive sites. Furthermore, small particles can be embedded into porous polymeric structures without the disadvantages of classical chromatographic columns (high pressure drop, fouling and plugging sensitivity, low flow rate), but with the advantages of membrane technology (easy scale-up, low-pressure drop, high flow rate). The adsorptive membranes feature high static as well as dynamic protein adsorption capacities for operating flow rates ranging from 200 to 400 L h bar per m(2) and ionic strength of 20-200 mM. In a sequential desorption step by changing the pH and/or the ionic strength of the eluent, up to 90% protein recovery was obtained. Next to the separation, the mixed matrix adsorber membrane functions as a concentration medium since the protein can be concentrated up to 20-fold in the eluent. The adsorber membranes can be reused in multiple adsorption/desorption cycles with good adsorption performances.     

Lipase adsorption as a factor of lipolysis regulation

Sorption isotherms of pancreatic lipase on solid supports were studied. It was shown that the enzyme adsorption can be described by Langmuir equation for hydrophobic surface and by the equation which takes into account reversible dimerization of the protein in the absorption layer for hydrophilic surface. The catalytic properties of adsorbed lipase depend on the nature of solid support. The significant role of the structure of adsorption layer in heterogeneous activation of the enzyme on hydrophobic surface was suggested.     

Quantification of streptavidin adsorption in microtitration wells

Streptavidin-coated microtitration plates have an important role as a solid phase in clinical diagnostics. We have designed techniques for evaluating quantitative and functional aspects of streptavidin adsorbed in microtitration wells. The theoretical monolayer adsorption capacity was modeled based on the molecular dimensions of the protein. Adsorbed streptavidin was quantified by direct labeling of protein with terbium chelate and with a sensitive bicinchoninic acid-based protein assay. A new small molecular weight (1037Da) reporter molecule, a europium-labeled biotin (Eu-biotin), was synthesized and used for monitoring adsorption and for determination of biotin-binding capacities of the streptavidin-coated wells. The theoretical monolayer adsorption of streptavidin yielded 6.20 pmol/cm(2) (370 ng) and consequently the theoretical adsorption capacity of a C12-format microtitration well (200 microl liquid, coated area 1.54 cm(2)) was 9.55 pmol/well (570 ng). Adsorption properties of streptavidin from two suppliers were tested, one of which yielded 350-380 ng/well while the other yielded over 500 ng/well. The biotin binding capacities were about 11 and 14 pmol/well, respectively. We managed to quantify surface-adsorbed streptavidin with sensitive fluorescence and protein measurement methods in the microtitration well. The new Eu-biotin reporter molecule enabled an exact and convenient determination of the biotin-binding capacities of streptavidin surfaces.     

Biothermodynamic analysis of BSA adsorption to alum gel using isothermal titration calorimetry

In this study, we used ITC (isothermal titration calorimetry) to quantitatively investigate the impacts of temperature and protein concentration on adsorption behavior on a solid surface, using BSA (bovine serum albumin) as a model protein, and alum (aluminum hydroxide) gel as an adsorbent. The zeta potential measurement for alum gel (0.25 mV at pH 9.3) revealed that its surface charge was not strong enough for electrostatic interaction. ITC analysis showed that the BSA-alum gel interaction was entropy-driven, suggesting that during adsorption, water molecules were expelled from the hydration layers of the alum gel and BSA. Therefore, the major mechanism for the BSA-alum gel interaction was hydrophobic interaction rather than electrostatic interaction. This biothermodynamic approach can be helpful not only to identify interaction mechanisms, but also to explore the optimum conditions for protein-adsorbent interactions.     

Modeling and simulation of liquid–solid circulating fluidized bed ion exchange system for continuous protein recovery

Liquid–solid circulating fluidized bed (LSCFB) is an integrated two‐column (downcomer and riser) system which can accommodate two separate processes (adsorption and desorption) in the same unit with continuous circulation of the solid particles between the two columns. In this study, a mathematical model based on the assumption of homogeneous fluidization was developed considering hydrodynamics, adsorption‐desorption kinetics and liquid–solid mass transfer. The simulation results showed good agreement with the available experimental results for continuous protein recovery. A parametric sensitivity study was performed to better understand the influence of different operating parameters on the BSA adsorption and desorption capacity of the system. The model developed can easily be extended to other applications of LSCFB. Biotechnol. Bioeng. 2009; 104: 111–126 © 2009 Wiley Periodicals, Inc.     

Mobility of adsorbed proteins: a Brownian dynamics study

We simulate the adsorption of lysozyme on a solid surface, using Brownian dynamics simulations. A protein molecule is represented as a uniformly charged sphere and interacts with other molecules through screened Coulombic and double-layer forces. The simulation starts from an empty surface and attempts are made to introduce additional proteins at a fixed time interval that is inversely proportional to the bulk protein concentration. We examine the effect of ionic strength and bulk protein concentration on the adsorption kinetics over a range of surface coverages. The structure of the adsorbed layer is examined through snapshots of the configurations and quantitatively with the radial distribution function. We extract the surface diffusion coefficient from the mean square displacement. At high ionic strengths the Coulombic interaction is effectively shielded, leading to increased surface coverage. This effect is quantified with an effective particle radius. Clustering of the adsorbed molecules is promoted by high ionic strength and low bulk concentrations. We find that lateral protein mobility decreases with increasing surface coverage. The observed trends are consistent with previous theoretical and experimental studies.     

Protein separation using membrane-encapsulated soluble ligand conjugates.

A new approach for isolating and recovering biological macromolecules using membrane-encapsulated soluble ligand conjugates was investigated. Membrane-encapsulated solid adsorbents have been successfully developed and employed in our laboratory to isolate and purify proteins and enzymes directly from culture broths. This new concept also makes it possible to use soluble ligand conjugates instead of solid adsorbents inside membrane capsules. In this work, model membrane-encapsulated soluble and insoluble ligands comprising Blue Dextran and Blue Sepharose entrapped within calcium alginate membranes were studied to compare adsorption characteristics such as capacities and rates. Experimental results suggest that membrane-encapsulated soluble ligands may be expected to result in higher overall adsorption capacity compared to membrane-encapsulated solid adsorbents with comparable adsorption rates.     

The continuous isolation of trypsin by affinity adsorbent recycling

A method for the continuous affinity separation of proteins is described in which the adsorbent, in the form of a polymer belt, is recycled through feedstock and eluent liquid flows. As the belt is nonporous, contact between the solute and the ligand is not diffusion-dependent. Consequently, rapid cycle rates are possible. Soybean trypsin inhibitor immobilized on nylon was used as an affinity ligand for the isolation of trypsin. During a 30-h continuous run, trypsin was isolated from a crude preparation of bovine pancreas with a recovery of 30% to 40%. Approximately 18 mg of trypsin was obtained from 500 mg of protein using a total of approximately 10 mug of ligand. Electrophoretic analysis of the eluent showed that chymotrypsin, which also binds to SBTI, was the only major contaminant of the product. It was demonstrated that the highest rates of protein purification were obtained using solid/liquid contact times well below that required to achieve saturation of the affinity adsorbent. Slower adsorbent recycle rates, which achieved higher protein binding per unit area of belt, resulted in lower protein purification per unit time. The rate of purification was also dependent on the concentration of target protein in the adsorption chamber at steady state. As high concentrations increased losses from the chamber outflow, this resulted in a compromise between throughput and recovery during the adsorption phase. Under the conditions investigated, recoveries of over 60% were obtained, and a maximum throughput of approximately 2.5 mg trypsin per hour was achieved. Preliminary studies have shown that this can be improved by compartmentalizing the adsorption chamber, which can reduce losses from the adsorption chamber to less than 5%. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 538-545, 1997.     

Interfacial adsorption of antifreeze proteins: a neutron reflection study

Interfacial adsorption from two antifreeze proteins (AFP) from ocean pout (Macrozoarces americanus, type III AFP, AFP III, or maAFP) and spruce budworm (Choristoneura fumiferana, isoform 501, or cfAFP) were studied by neutron reflection. Hydrophilic silicon oxide was used as model substrate to facilitate the solid/liquid interfacial measurement so that the structural features from AFP adsorption can be examined. All adsorbed layers from AFP III could be modeled into uniform layer distribution assuming that the protein molecules were adsorbed with their ice-binding surface in direct contact with the SiO₂ substrate. The layer thickness of 32 Å was consistent with the height of the molecule in its crystalline form. With the concentration decreasing from 2 mg/ml to 0.01 mg/ml, the volume fraction of the protein packed in the monolayer decreased steadily from 0.4 to 0.1, consistent with the concentration-dependent inhibition of ice growth observed over the range. In comparison, insect cfAFP showed stronger adsorption over the same concentration range. Below 0.1 mg/ml, uniform layers were formed. But above 1 mg/ml, the adsorbed layers were characterized by a dense middle layer and two outer diffuse layers, with a total thickness around 100 Å. The structural transition indicated the responsive changes of conformational orientation to increasing surface packing density. As the higher interfacial adsorption of cfAFP was strongly correlated with the greater thermal hysteresis of spruce budworm, our results indicated the important relation between protein adsorption and antifreeze activity.