Hollow-fiber flow field-flow fractionation with multi-angle laser scattering detection for aggregation studies of therapeutic proteins.

The rapid development of protein-based pharmaceuticals highlights the need for robust analytical methods to ensure their quality and stability. Among proteins used in pharmaceutical applications, an important and ever increasing role is represented by monoclonal antibodies and large proteins, which are often modified to enhance their activity or stability when used as drugs. The bioactivity and the stability of those proteins are closely related to the maintenance of their complex structure, which however are influenced by many external factors that can cause degradation and/or aggregation. The presence of aggregates in these drugs could reduce their bioactivity and bioavailability, and induce immunogenicity. The choice of the proper analytical method for the analysis of aggregates is fundamental to understand their (size) dimensional range, their amount, and if they are present in the sample as generated by an aggregation or as an artifact due to the method itself. Size exclusion chromatography is one of the most important techniques for the quality control of pharmaceutical proteins; however, its application is limited to relatively low molar mass aggregates. Among the techniques for the size characterization of proteins, field-flow fractionation (FFF) represents a competitive choice because of its soft mechanism due to the absence of a stationary phase and application in a broader size range, from nanometer- to micrometer-sized analytes. In this paper, the microcolumn variant of FFF, the hollow-fiber flow FFF, was online coupled with multi-angle light scattering, and a method for the characterization of aggregates with high reproducibility and low limit of detection was demonstrated employing an avidin derivate as sample model.

Generation of a miniaturized free-flow electrophoresis chip based on a multi-lamination technique--isoelectric focusing of proteins and a single-stranded DNA fragment.

Free-flow electrophoresis techniques have been applied for separations in various areas of chemistry and biochemistry. Here we focus on the generation of a free-flow electrophoresis chip and direct monitoring of the separation of different molecules in the separation bed of the miniaturized chip. We demonstrate a fast and efficient way to generate a low-cost micro-free-flow electrophoresis (µFFE) chip with a filling capacity of 9.5 µL based on a multi-lamination technique. Separating webs realized by two transfer-adhesive tapes avoid the problem of gas bubbles entering the separation area. The chip is characterized by isoelectric focusing markers (IEF markers). The functionality of the chip is demonstrated by free-flow isoelectric focusing (FFIEF) of the proteins BSA (bovine serum albumin) and avidin and a single-stranded DNA (ssDNA) fragment in the pH range 3 to 10. The separation voltage ranges between 167 V cm(-1) and 422 V cm(-1), depending on the application.

Simultaneous optical and electrochemical label-free biosensing with ITO-coated lossy-mode resonance sensor.

In this work we discuss a new label-free biosensing device based on indium tin oxide (ITO) overlaid section of a multimode optical fiber fused silica core. The sensor has been used to optical measurements also simultaneously interrogated electrochemically (EC). Due to optimized thickness and optical properties of ITO film, a lossy-mode resonance (LMR) could be observed in the optical domain, where electrical properties of the film allowed for application of the sensor as a working electrode in an EC setup. It has been confirmed that the LMR response depends on optical properties of the external medium, as well as potential applied to the electrode during cyclic voltammetry. After the ITO surface functionalization with amine groups and covalently attached biotin, the device has been applied for label-free biosensing of avidin in both the domains simultaneously. On the example of biotin-avidin detection system it was demonstrated that when avidin concentration increases a decrease in current and increase in LMR wavelength shift were recorded in EC and optical domain, respectively. Both optical and EC responses follow the protein interaction process, and thus can be used as cross-verification of the readouts. Moreover, an extended information has been achieved comparing to solely EC interrogation, i.e., the grafting process of biotin and avidin was directly monitored optically displaying individual steps of an incubation procedure.

A suitable and effective stepwise oxidative refolding procedure for highly-cationic tetrameric avidin in nucleic acid free conditions.

Optimized conditions are needed to refold recombinant proteins from bacterial inclusion bodies into their biologically active conformations. In this study, we found two crucial requirements for efficient refolding of cationic tetrameric chicken avidin. The first step is to eliminate nucleic acid contaminants from the bacterial inclusion body. The electrostatic interactions between the remaining nucleic acids and proteins strongly enhanced protein aggregation during the refolding process. The cysteine specific reversible S-cationization procedure was successfully employed for large-scale preparation of nucleic acid free denatured protein without purification tag system. The second step is the intramolecular disulfide formation prior to refolding in dialysis removing denaturant. Disulfide intact monomeric avidin showed efficient formation of biologically active tetrameric conformation during the refolding process. Using this optimized refolding procedure, highly cationic avidin derivative designed as an intracellular delivery carrier of biotinylated protein was successfully prepared.

Ultrasensitive tantalum oxide nano-coated long-period gratings for detection of various biological targets.

In this work we discussed a label-free biosensing application of long-period gratings (LPGs) optimized in refractive index (RI) sensitivity by deposition of thin tantalum oxide (TaOx) overlays. Comparing to other thin film and materials already applied for maximizing the RI sensitivity, TaOx offers good chemical and mechanical stability during its surface functionalization and other biosensing experiments. It was shown theoretically and experimentally that when RI of the overlay is as high as 2 in IR spectral range, for obtaining LPGs ultrasensitive to RI, the overlay's thickness must be determined with subnanometer precision. In this experiment the TaOx overlays were deposited using Atomic Layer Deposition method that allowed for achieving overlays with exceptionally well-defined thickness and optical properties. The TaOx nano-coated LPGs show RI sensitivity determined for a single resonance exceeding 11,500 nm/RIU in RI range nD= 1.335-1.345 RIU, as expected for label-free biosensing applications. Capability for detection of various in size biological targets, i.e., proteins (avidin) and bacteria (Escherichia coli), with TaOx-coated LPGs was verified using biotin and bacteriophage adhesin as recognition elements, respectively. It has been shown that functionalization process, as well as type of recognition elements and target analyte must be taken into consideration when the LPG sensitivity is optimized. In this work optimized approach made possible detection of small in size biological targets such as proteins with sensitivity reaching 10.21 nm/log(ng/ml).

Hydrophobin (HFBI): A potential fusion partner for one-step purification of recombinant proteins from insect cells.

Hydrophobins play an important role in binding and assembly of fungal surface structures as well as in medium-air interactions. These, hydrophobic properties provide interesting possibilities when purification of macromolecules is concerned. In aqueous micellar two-phase systems, based on surfactants, the water soluble hydrophobins are concentrated inside micellar structures and, thus, distributed to defined aqueous phases. This, one-step purification is attractive particularly when large-scale production of recombinant proteins is concerned. In the present study the hydrophobin HFBI of Trichoderma reesei was expressed as an N-terminal fusion with chicken avidin in baculovirus infected insect cells. The intracellular distribution of the recombinant fusion construct was analyzed by confocal microscopy and the protein subsequently purified from cytoplasmic extracts in an aqueous micellar two-phase system by using a non-ionic surfactant. The results show that hydrophobin and an avidin fusion thereof were efficiently expressed in insect cells and that these hydrophobic proteins could be efficiently purified from these cells in one-step by adopting an aqueous micellar two-phase system.

Affinity purification of the avidin protein family, based on crystal structures of avidin-HABA complexes.

Since the importance of the high affinity between avidin and biotin, Kd = 3 × 10-16 M, gained universal recognition, numerous chemical, biological and medical avidin-biotin based applications have been developed. However, in some cases the high affinity may be a disadvantage, as this interaction is irreversible under physiological conditions. The dye, 4'-hydroxyazobenzene-2-carboxylic acid (HABA), binds avidin, at the biotin binding site, as determined by X-ray, at a much lower affinity constant, Kd = 6 × 10-6 M. We prepared a HABA affinity column (amber colored). Avidin bound to the column at a pH between 4 and 8.5, causing a change of color to red, and it could be eluted at mild conditions with buffers containing biotin, HABA, 1.5 M potassium chloride or a pH lower than 4.0 or higher than 8.5. Avidin eluted with HABA, created a red avidin-HABA complex, which was visualized. HABA free avidin was obtained by dialysis, which was followed by the loss of red coloration. The novel and easy to use HABA-affinity column was employed in our lab to prepare pure, fully glycosylated avidin from egg white. Most importantly, it may serve as an ideal tool for educational purposes, illuminating concepts of molecular recognition, reversible molecular binding, structure-based molecular design and solid phase chemical synthesis, as it is a reliable and visible reagent.

Controlling quaternary structure assembly: subunit interface engineering and crystal structure of dual chain avidin.

Dual chain avidin (dcAvd) is an engineered avidin form, in which two circularly permuted chicken avidin monomers are fused into one polypeptide chain. DcAvd can theoretically form two different pseudotetrameric quaternary assemblies because of symmetry at the monomer-monomer interfaces. Here, our aim was to control the assembly of the quaternary structure of dcAvd. We introduced the mutation I117C into one of the circularly permuted domains of dcAvd and scanned residues along the 1-3 subunit interface of the other domain. Interestingly, V115H resulted in a single, disulfide locked quaternary assembly of dcAvd, whereas I117H could not guide the oligomerisation process even though it stabilised the protein. The modified dcAvd forms were found to retain their characteristic pseudotetrameric state both at high and low pH, and were shown to bind D-biotin at levels comparable to that of wild-type chicken avidin. The crystal structure of dcAvd-biotin complex at 1.95 Angstroms resolution demonstrates the formation of the functional dcAvd pseudotetramer at the atomic level and reveals the molecular basis for its special properties. Altogether, our data facilitate further engineering of the biotechnologically valuable dcAvd scaffold and gives insights into how to guide the quaternary structure assembly of oligomeric proteins.

Factors dictating the pseudocatalytic efficiency of avidins.

The hydrolysis of biotinyl p-nitrophenyl ester (BNP) by a series of avidin derivatives was examined. Surprisingly, a hyperthermostable avidin-related protein (AVR4) was shown to display extraordinary yet puzzling hydrolytic activity. In order to evaluate the molecular determinants that contribute to the reaction, the crystal structure of AVR4 was compared with those of avidin, streptavidin and key mutants of the two proteins in complex with biotinyl p-nitroanilide (BNA), the inert amide analogue of BNP. The structures revealed that a critical lysine residue contributes to the hydrolysis of BNP by avidin but has only a minor contribution to the AVR4-mediated reaction. Indeed, the respective rates of hydrolysis among the different avidins reflect several molecular parameters, including binding-site architecture, the availability of the ligand to solvent and the conformation of the ligand and consequent susceptibility to efficient nucleophilic attack. In avidin, the interaction of BNP with Lys111 and disorder of the L3,4 loop (and consequent solvent availability) together comprise the major driving force behind the hydrolysis, whereas in AVR4 the status of the ligand (the pseudo-substrate) is a major distinguishing feature. In the latter protein, a unique conformation of the L3,4 loop restrains the pseudo-substrate, thereby exposing the carbonyl carbon atom to nucleophilic attack. In addition, due to its conformation, the pseudo-substrate in the AVR4 complex cannot interact with the conserved lysine analogue (Lys109); instead, this function is superseded by polar interactions with Arg112. The results demonstrate that, in highly similar proteins, different residues can perform the same function and that subtle differences in the active-site architecture of such proteins can result in alternative modes of reaction.

Immobilized sialyltransferase fused to a fungal biotin-binding protein: Production, properties, and applications.

A ß-galactoside α2,6-sialyltransferase (ST) from the marine bacterium Photobacterium sp. JT-ISH-224 with a broad acceptor substrate specificity was fused to a fungal biotin-binding protein tamavidin 2 (TM2) to produce immobilized enzyme. Specifically, a gene for the fusion protein, in which ST from Photobacterium sp. JT-ISH-224 and TM2 were connected via a peptide linker (ST-L-TM2) was constructed and expressed in Escherichia coli. The ST-L-TM2 was produced in the soluble form with a yield of approximately 15,000 unit/300 ml of the E. coli culture. The ST-L-TM2 was partially purified and part of it was immobilized onto biotin-bearing magnetic microbeads. The immobilized ST-L-TM2 onto microbeads could be used at least seven consecutive reaction cycles with no observed decrease in enzymatic activity. In addition, the optimum pH and temperature of the immobilized enzyme were changed compared to those of a free form of the ST. Considering these results, it was strongly expected that the immobilized ST-L-TM2 was a promising tool for the production of various kind of sialoligosaccharides.

Dual-affinity avidin molecules.

A recently reported dual-chain avidin was modified further to contain two distinct, independent types of ligand-binding sites within a single polypeptide chain. Chicken avidin is normally a tetrameric glycoprotein that binds water-soluble d-biotin with extreme affinity (K(d) approximately 10(-15) M). Avidin is utilized in various applications and techniques in the life sciences and in the nanosciences. In a recent study, we described a novel avidin monomer-fusion chimera that joins two circularly permuted monomers into a single polypeptide chain. Two of these dual-chain avidins were observed to associate spontaneously to form a dimer equivalent to the wt tetramer. In the present study, we successfully used this scaffold to generate avidins in which the neighboring biotin-binding sites of dual-chain avidin exhibit two different affinities for biotin. In these novel avidins, one of the two binding sites in each polypeptide chain, the pseudodimer, is genetically modified to have lower binding affinity for biotin, whereas the remaining binding site still exhibits the high-affinity characteristic of the wt protein. The pseudotetramer (i.e., a dimer of dual-chain avidins) has two high and two lower affinity biotin-binding sites. The usefulness of these novel proteins was demonstrated by immobilizing dual-affinity avidin with its high-affinity sites. The sites with lower affinity were then used for affinity purification of a biotinylated enzyme. These "dual-affinity" avidin molecules open up wholly new possibilities in avidin-biotin technology, where they may have uses as novel bioseparation tools, carrier proteins, or nanoscale adapters.

Localization of avidin in virus-transformed and damaged chicken embryo fibroblasts by immunofluorescence and the avidin-biotin-peroxidase complex (ABC) technique.

Avidin, a high-affinity biotin-binding protein of chicken oviduct, was recently found to be synthesized and secreted by damaged or virus-transformed chicken embryo fibroblasts and by chicken macrophages. We have now localized avidin in fibroblasts that were transformed by Rous sarcoma virus. The cells released to the culture medium up to 12 micrograms avidin per 10(6) cells, as judged by the [14C] biotin-binding method. In immunofluorescence microscopy, avidin was localized to the cytoplasm of transformed and of untransformed damaged cells. Treatment with the ionophore monensin was used to determine whether avidin is processed through the Golgi region, which was localized using rhodamine-labeled wheat germ agglutinin. Under these conditions avidin was largely confined to the Golgi region. At the electron microscopic level avidin could be localized to the endoplasmic reticulum of transformed cells, using anti-avidin antibodies and the avidin-biotin-peroxidase complex (ABC) technique. Biotinyl peroxidase did not stain the endogenous avidin in cell layers processed for light or electron microscopy indicating that its biotin-binding sites were either saturated or denaturated. The possibility that endogenous avidin in tissues or cell cultures may bind biotinylated reagents should be controlled for in techniques involving the avidin-biotin interaction.

Partial purification of a progesterone-inducible messenger RNA (avidin) from hen oviduct.

The messenger RNA (mRNA) for avidin, which represents less than 0.05% of the total cellular proteins, was partially purified from hen oviduct, and the presence of avidin mRNA was shown to depend upon prior stimulation by progesterone. A total nucleic acid extract was subjected to oligo (dT)-cellulose chromatography, followed by Sepharose 4B chromatography, preparative agarose gel electrophoresis, and sucrose gradient centrifugation. The relative purity of each preparation was assessed by translation in a wheat-germ system; avidin messenger RNA activity was measured by specific immunoprecipitation of synthesized proteins. Avidin mRNA was separated from the bulk of the total messenger RNA activity of the oviduct and from all ribosomal RNAs to produce greater than a 1000-fold enrichment of avidin mRNA activity compared with total cellular RNA. Based on the translation assay, the most highly purified fraction contained about 2.5% avidin messenger RNA. Avidin mRNA activity was absent in partially purified mRNA obtained from estrogen-stimulated chick oviducts, but was detected in oviducts following progesterone administration.

Recombinant NeutraLite avidin: a non-glycosylated, acidic mutant of chicken avidin that exhibits high affinity for biotin and low non-specific binding properties.

A recombinant non-glycosylated and acidic form of avidin was designed and expressed in soluble form in baculovirus-infected insect cells. The mutations were based on the same principles that guided the design of the chemically and enzymatically modified avidin derivative, known as NeutraLite Avidin. In this novel recombinant avidin derivative, five out of the eight arginine residues were replaced with neutral amino acids, and two of the lysine residues were replaced by glutamic acid. In addition, the carbohydrate-bearing asparagine-17 residue was altered to an isoleucine, according to the known sequences of avidin-related genes. The resultant mutant protein, termed recombinant NeutraLite Avidin, exhibited superior properties compared to those of avidin, streptavidin and the conventional NeutraLite Avidin, prepared by chemo-enzymatic means. In this context, the recombinant mutant is a single molecular species, which possesses strong biotin-binding characteristics. Due to its acidic pI, it is relatively free from non-specific binding to DNA and cells. The recombinant NeutraLite Avidin retains seven lysines per subunit, which are available for further conjugation and derivatization.

Determination of biotin on a protein by quantitative sodium dodecyl sulfate-capillary gel electrophoresis of monomeric avidin.

Sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) is performed to quantify monomeric avidin and biotin on a protein. Under non-reducing SDS-CGE conditions, avidin migrates as monomers exhibiting apparent molecular mass 17,000. In the presence of a biotin-protein conjugate, monomeric avidin binds the conjugate and forms a larger complex that migrates later in the separation. The difference between the remaining monomeric avidin and the initial amount is the portion of monomeric avidin bound to the conjugate. Accordingly, the number of biotin on the protein can be calculated. The assay is linearly responsive to increasing biotin loading in a biotinylation reaction of a protein. Accuracy of the assay is also demonstrated by good sample dilution recovery. Excellent quantitative reproducibility < 2% (relative standard deviation) is obtained for both intra- and inter-day measurements. Main advantages of the method include the use of monomeric avidin that minimizes steric hindrance to capture biotin on a protein and assay automation on a capillary electrophoresis apparatus.

N-laurylbiotinamide as affinity surfactant.

N-laurylbiotinamide (NLB), which retains strong affinity for the protein avidin, was synthesized from biotin and N-laurylamine via the biotin ester of N-hydroxysuccinimide and characterized by NMR. When the synthesized NLB was used as a cosurfactant with AOT to form a reverse micellar system in isooctane, it was found to extend the pH range over which avidin can be transferred from a continuous aqueous solution to the reverse-micellar phase. This behavior is similar to that already reported for a different affinity surfactant, n-octyl beta-D-glucopyranoside.

Affinity purification of lipid vesicles.

We present a novel column chromatography technique for recovery and purification of lipid vesicles, which can be extended to other macromolecular assemblies. This technique is based on reversible binding of biotinylated lipids to monomeric avidin. Unlike the very strong binding of biotin and biotin-functionalized molecules to streptavidin, the interaction between biotin-functionalized molecules and monomeric avidin can be disrupted effectively by ligand competition from free biotin. In this work, biotin-functionalized lipids (biotin-PEG-PE) were incorporated into synthetic lipid vesicles (DOPC), resulting in unilamellar biotinylated lipid vesicles. The vesicles were bound to immobilized monomeric avidin, washed extensively with buffer, and eluted with a buffer supplemented with free biotin. Increasing the biotinyl lipid molar ratio beyond 0.53% of all lipids did not increase the efficiency of vesicle recovery. A simple adsorption model suggests 1.1 x 10(13) active binding sites/mL of resin with an equilibrium binding constant of K = 1.0 x 10(8) M(-1). We also show that this method is very robust and reproducible and can accommodate vesicles of varying sizes with diverse contents. This method can be scaled up to larger columns and/or high throughput analysis, such as a 96-well plate format.

Use of water-dispersible Fe(2)O(3) nanoparticles with narrow size distributions in isolating avidin.

Synthetic approaches that vigorously control the microstructures of water-dispersible gamma-Fe(2)O(3) nanoparticles such as size and size uniformity are of importance to the potential biological applications of these nanomaterials. In the present paper, water-dispersible gamma-Fe(2)O(3) nanocrystals with narrow size distributions (bipy-Fe(2)O(3)) were prepared via a site-exchange reaction. These particular materials are superparamagnetic and stable within a wide range of pH. Introduction of the biotin functionality onto the surfaces of bipy-Fe(2)O(3) enabled the affinity isolation of the protein avidin from its incubation solution magnetically with 96% efficiency.

Preparation of deglycosylated egg white avidin.

A simple procedure for the preparation of deglycosylated avidin is described. Commercially obtained avidin was treated with a mixed microbial culture. The cells were capable of growing on the oligosaccharide residues, but generally ignored the polypeptide portion of the egg white glycoprotein. The resultant deglycosylated avidin retained its biotin-binding characteristics. The major bacterial strain (strain BECH080), responsible for the deglycosylation, was isolated. On the basis of elementary biochemical tests, fatty acid, and phenotypic analyses, the isolate was identified as a strain of Flavobacterium meningosepticum. The primary enzymatic activity that caused the removal of the oligosaccharide residues of avidin appeared to be similar to endoglycosidase F.

[Irreversible chemical AFM-fishing for the detection of low-copied proteins].

The atomic-force microscopy-based method of irreversible chemical AFM-fishing (AFM-IF(Ch)) has been developed for the detection of proteins at ultra-low concentrations in solution. Using this method, a very low concentration of horseradish peroxidase (HRP) protein (10(-17) M) was detected in solution. A theoretical model that allows the description of obtained experimental data, is proposed. This model takes into consideration both the transport of the protein from the bulk solution onto the AFM-chip surface and its irreversible binding to the activated area.