Pure Protein Solutions Research Articles

Hydrophilic modification of P(VDF- co-CTFE) porous membranes

This paper describes fabrication of a poly(vinylidene difluoride- co-chlorotrifluoroethylene) (P(VDF- co-CTFE)) porous membrane via non-solvent induced phase inversion and subsequent hydrophilic modification using high efficient surface initiated atom transfer radical polymerization (ATRP). The effect of viscosities of casting solutions on microstructures of the P(VDF- co-CTFE) membrane was investigated. The surface chemistry, thermal stability, morphological structure, and hydrophilicity of the modified membranes were evaluated by Fourier Transform Infrared Attenuated Total Reflection (FTIR-ATR), Differential Scanning Calorimeter (DSC), Scanning Electron Microscope (SEM), and contact angle measurements, respectively. The degree of grafting and the degree of swelling were measured to analyze the effect of polymerization time on the wettability. The mechanical strength of the membranes after modification was also investigated. The permeability and fouling resistance were evaluated according to pure water flux and protein solution filtration measurements. The results demonstrate that the hydrophobic P(VDF- co-CTFE) membrane can be feasibly modified by immobilization of hydrophilic poly(ethylene glycol) methyl ether methacrylate (PEGMA) brushes via surface initiated ATRP.

Integrated solution to purification challenges in the manufacture of a soluble recombinant protein in E. coli

Apolipoprotein A 1 Milano (ApoA-1M), the protein component of a high-density lipoprotein (HDL) mimic with promising potential for reduction of atherosclerotic plaque, is produced at large scale by expression in E. coli. Significant difficulty with clearance of host cell proteins (HCPs) was experienced in the original manufacturing process despite a lengthy downstream purification train. Analysis of purified protein solutions and intermediate process samples led to identification of several major HCPs co-purifying with the product and a bacterial protease potentially causing a specific truncation of ApoA-1M found in the final product. Deletion of these genes from the original host strain succeeded in substantially reducing the levels of HCPs and the truncated species without adversely affecting the overall fermentation productivity, contributing to a much more efficient and robust new manufacturing process.

Droplet Evaporation of Pure Water and Protein Solution on Nanostructured Superhydrophobic Surfaces of Varying Heights

Evaporation of liquids on substrates is important for many applications including lab-on-a-chip, especially when they are in droplets. Unlike on planar substrates, droplet evaporation on micropatterned substrates has been studied only recently and none so far on nanopatterns. Driven by the applicability of nanostructured surfaces to biomaterials and tissue engineering, we report on the evaporative process of sessile droplets of pure water and a protein solution on superhydrophobic surfaces of sharp-tip post structures in a submicrometer pitch (230 nm) and varying heights (100-500 nm). We find that the nanotopographical three-dimensionalities such as structural height and sidewall profile affect the surface superhydrophobicity in such a way that only tall and slender nanostructures provide the surface with great superhydrophobicity (a contact angle more than 170 degrees). The evaporation process was different between the pure water and the protein solution; unlike pure water, a significant contact-line spreading and pinning effect was observed in a droplet of a protein solution with an intermediate transition from a dewetting (Cassie) to a wetting (Wenzel) state. Enabled by well-defined nanostructures, our results highlight that the surface superhydrophobicity and the droplet evaporation are significantly affected by the three-dimensional nanometric topography and the surface fouling such as protein adsorption.

Production of biohybrid protein/PEO scaffolds by electrospinning

AbstractElectrospun polymeric micro‐ and nanofibers offer the opportunity to reproduce the filamentous structure of the extracellular matrix in native tissues. The ability to electrospin extracellular matrix proteins offers the additional benefit of being able to create a chemical and structural microenvironment for cells in order to influence their behavior during scaffold seeding and tissue formation. This offers significant advantages over unspecific protein adsorption or functionalisation with adhesion promoting peptide sequences. However, producing electrospun fibers from pure protein solutions remains difficult and costly. A more accessible approach is to introduce proteins into electrospun fibers by blending synthetic polymer solutions with selected extracellular matrix proteins. The correct choice of polymer also makes the usage of toxic and denaturing solvents unnecessary. In this study we have investigated the production process for and the suitability of protein/PEO fibers, specifically collagen I/PEO, as cell culture substrates.

Preparation and characterization of polysulfone affinity membranes bearing a synthetic peptide ligand for the separation of murine immunoglobulins

Affinity membranes have been prepared by immobilizing D-PAM, a synthetic ligand that exhibits affinity for the Fc portion of antibodies, onto poliethersulfone microporous membranes. The ligand density has been measured and the ligand utilization was evaluated and compared with literature data available for chromatographic beads. The resulting new affinity membranes have been experimentally characterized and tested by using pure murine IgG solutions and mouse serum. Equilibrium and kinetic parameters have been obtained in batch experiments using pure protein solutions. The highest binding capacity measured for murine IgG was 45 microg/cm(2) obtained at 1.2 mg/mL protein concentration at equilibrium, while the maximum static binding capacity calculated with the Langmuir model was 81 microg/cm(2). The adsorption of murine IgG on the affinity membranes was described using different isotherms: Freundlich and Temkin models have been considered and critically compared with the Langmuir adsorption model. A dynamic binding capacity of 21 microg/cm(2) was obtained by feeding a solution of 0.3 mg/mL of murine IgG, confirming the results obtained in batch experiments at the same concentration. The affinity membranes considered are endowed with good binding capacity for murine IgG and good selectivity for immunoglobulins and can be considered for the capturing step of an antibody production process.

Structure of the Bacteriophage φKZ Lytic Transglycosylase gp144

Lytic transglycosylases are enzymes that act on the peptidoglycan of bacterial cell walls. They cleave the glycosidic linkage between N-acetylmuramoyl and N-acetylglucosaminyl residues with the concomitant formation of a 1,6-anhydromuramoyl product. The x-ray structure of the lytic transglycosylase gp144 from the Pseudomonas bacteriophage phi KZ has been determined to 2.5-A resolution. This protein is probably employed by the bacteriophage in the late stage of the virus reproduction cycle to destroy the bacterial cell wall to release the phage progeny. phi KZ gp144 is a 260-residue alpha-helical protein composed of a 70-residue N-terminal cell wall-binding domain and a C-terminal catalytic domain. The fold of the N-terminal domain is similar to the peptidoglycan-binding domain from Streptomyces albus G D-Ala-D-Ala carboxypeptidase and to the N-terminal prodomain of human metalloproteinases that act on extracellular matrices. The C-terminal catalytic domain of gp144 has a structural similarity to the catalytic domain of the transglycosylase Slt70 from Escherichia coli and to lysozymes. The gp144 catalytic domain has an elongated groove that can bind at least five sugar residues at sites A-E. As in other lysozymes, the peptidoglycan cleavage (catalyzed by Glu 115 in gp144) occurs between sugar-binding subsites D and E. The x-ray structure of the phi KZ transglycosylase complexed with the chitotetraose (N-acetylglucosamine)(4) has been determined to 2.6-A resolution. The N-acetylglucosamine residues of the chitotetraose bind in sites A-D.

Reversible and irreversible membrane fouling during in-line microfiltration of concentrated protein solutions

In-line microfiltration which is widely used for sterilization of therapeutic proteins prior to formulation is frequently operated at constant flux by pumping purified protein solution through a membrane. Increase in transmembrane pressure (TMP) due to membrane fouling is a major problem in such processes. This study examines how resistance to filtration increases during in-line microfiltration of concentrated protein solutions. The TMP was found to increase in three distinct phases, each characterized by its own rate and extent of reversibility. The major portion of the TMP increase was found to be reversible upon front washing with buffer without any kind of physical or chemical cleaning. There was no evidence of concentration polarization in the processes studied. Size exclusion chromatographic analysis of front wash samples suggested bovine serum albumin (BSA) monomer as the major foulant accountable for the reversible fouling. Aggregate free protein solutions prepared by pre-filtration showed negligible membrane fouling. We hypothesize that trace amounts of protein aggregates present in untreated feed solutions were responsible for initiating membrane fouling. However, the actual fouling was caused by the protein monomer which was sequestered on and within the membrane by association with the retained aggregates. The location of foulant on and within the membrane was examined by confocal laser scanning microscopy. The deposition of the foulant was found to be restricted to a zone very close to the membrane surface. Intermittent protein filtration experiments were carried out in an effort to gain better insight on the membrane fouling process and to identify new strategies to maximize product throughput.

Preparation and characterization of nanoparticles formed by chitosan–caseinate interactions

Intermacromolecular complexation between chitosan and sodium caseinate in aqueous solutions was studied as a function of pH (3–6.5), using absorbance measurements (at 600 nm), dynamic light scattering (DLS), and transmission electron microscopy (TEM). The chitosan–caseinate complexes formed were stable and soluble in the pH range 4.8–6.0. In this pH range, the biopolymers had opposite charges. At higher concentrations of chitosan (0.15 wt%), the soluble complexes associated to form larger particles. DLS data showed that, between pH 4.8 and 6.0, the particles formed by the complexation of chitosan and caseinate had sizes between 250 and 350 nm and these nanoparticles were visualized using negative staining TEM. Above pH 6.0, the nanoparticles associated to form larger particles, causing phase separation. Addition of NaCl increased the particle size. The pH dependence of the zeta potential of the mixture solutions was appreciably different from that of the pure protein and pure chitosan solutions.

Thermodynamic Consequences of Mutations in Vernier Zone Residues of a Humanized Anti-human Epidermal Growth Factor Receptor Murine Antibody, 528

To investigate the role of Vernier zone residues, which are comprised in the framework regions and underlie the complementarity-determining regions (CDRs) of antibodies, in the specific, high affinity interactions of antibodies with their targets, we focused on the variable domain fragment of murine anti-human epidermal growth factor receptor antibody 528 (m528Fv). Grafting of the CDRs of m528Fv onto a selected framework region of human antibodies, referred to as humanization, reduced the antibody's affinity for its target by a factor of 1/40. The reduction in affinity was due to a substantial reduction in the negative enthalpy change associated with binding. Crystal structures of the ligand-free antibody fragments showed no noteworthy conformational changes due to humanization, and the loop structures of the CDRs of the humanized antibodies were identical to those of the parent antibodies. Several mutants of the CDR-grafted (humanized) variable domain fragment (h528Fv), in which some of the Vernier zone residues in the heavy chain were replaced with the parental murine residues, were constructed and prepared using a bacterial expression system. Thermodynamic analyses of the interactions between the mutants and the soluble extracellular domain of epidermal growth factor receptor showed that several single mutations and a double mutation increased the negative enthalpy and heat capacity changes. Combination of these mutations, however, led to somewhat reduced negative enthalpy and heat capacity changes. The affinity of each mutant for the target was within the range for the wild-type h528Fv, and this similarity was due to enthalpy-entropy compensation. These results suggest that Vernier zone residues make enthalpic contributions to antigen binding and that the regulation of conformational entropy changes upon humanization of murine antibodies must be carefully considered and optimized.

Quartz crystal microbalance immunosensor for the detection of antibodies to double-stranded DNA

We report the development of a novel quartz crystal microbalance immunosensor with the simultaneous measurement of resonance frequency and motional resistance for the detection of antibodies to double-stranded DNA (dsDNA). The immobilization of poly(L-lysine) and subsequent complexation with DNA resulted in formation of a sensitive dsDNA-containing nanofilm on the surface of a gold electrode. Atomic force microscopy has been applied for the characterization of a poly(L-lysine)-DNA film. After the blocking with bovine serum albumin, the immunosensor in flow-injection mode was used to detect the antibodies to dsDNA in purified protein solutions of antibodies to dsDNA and to single-stranded DNA, monoclonal human immunoglobulin G, DNase I and in blood serum of patients with bronchial asthma and systemic lupus erythematosus. Experimental results indicate high sensitivity and selectivity of the immunosensor.

Surface immobilization of polymer brushes onto porous poly(vinylidene fluoride) membrane by electron beam to improve the hydrophilicity and fouling resistance

Poly(vinylidene fluoride) (PVDF) membrane was pre-irradiated by electron beam, and then poly(ethylene glycol) methyl ether methacrylate (PEGMA) was grafted onto the membrane surface in the aqueous solution. The degree of grafting was significantly influenced by the pH value of the reaction solution. The surface chemical changes were characterized by the Fourier transform infrared attenuated total reflection spectroscopy (FTIR-ATR) and X-ray photoelectron spectroscopy (XPS). Combining with the analysis of the nuclear magnetic resonance proton and carbon spectra ( 1H NMR and 13C NMR), PEGMA was mainly grafted onto the membrane surface. Morphological changes were characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The porosity and bulk mean pore size changes were determined by a mercury porosimeter. The surface and bulk hydrophilicity were evaluated on the basis of static water contact angle, dynamic water contact angle and the dynamic adsorption process. Furthermore, relative high permeation fluxes of pure water and protein solution were obtained. All these results demonstrate that both hydrophilicity and fouling resistance of the PVDF membrane can be improved by the immobilization of hydrophilic comb-like polymer brushes on the membrane surface.

RNA aptamers to mammalian initiation factor 4G inhibit cap-dependent translation by blocking the formation of initiation factor complexes

Eukaryotic translation initiation factor 4G (eIF4G) plays a crucial multimodulatory role in mRNA translation and decay by interacting with other translation factors and mRNA-associated proteins. In this study, we isolated eight different RNA aptamers with high affinity to mammalian eIF4G by in vitro RNA selection amplification. Of these, three aptamers (apt3, apt4, and apt5) inhibited the cap-dependent translation of two independent mRNAs in a rabbit reticulocyte lysate system. The cap-independent translation directed by an HCV internal ribosome entry site was not affected. Addition of exogenous eIF4G reversed the aptamer-mediated inhibition of translation. Even though apt3 and apt4 were selected independently, they differ only by two nucleotides. The use of truncated eIF4G variants in binding experiments indicated that apt4 (and probably apt3) bind to both the middle and C-terminal domains of eIF4G, while apt5 binds only to the middle domain of eIF4G. Corresponding to the difference in the binding sites in eIF4G, apt4, but not apt5, hindered eIF4G from binding to eIF4A and eIF3, in a purified protein solution system as well as in a crude lysate system. Therefore, the inhibition of translation by apt4 (and apt3) is due to the inhibition of formation of initiation factor complexes involving eIF4A and eIF3. On the other hand, apt5 had a much weaker affinity to eIF4G than apt4, but inhibited translation much more efficiently by an unknown mechanism. The five additional aptamers have sequences and predicted secondary structures that are largely different from each other and from apt3 through apt5. Therefore, we speculate that these seven sets of aptamers may bind to different regions in eIF4G in different fashions.

N-Linked Oligosaccharides on Chondroitin 6-Sulfotransferase-1 Are Required for Production of the Active Enzyme, Golgi Localization, and Sulfotransferase Activity toward Keratan Sulfate

We have shown previously that purified chondroitin 6-sulfotransferase-1 (C6ST-1) was a glycoprotein abundant in N-linked oligosaccharides and could sulfate both chondroitin (C6ST activity) and keratan sulfate (KSST activity); however, functional roles of the N-glycans have remained unclear. In the present study, we show essential roles of N-glycans attached to C6ST-1 in the generation of the active enzyme and in its KSST activity. Treatment with tunicamycin of COS-7 cells transfected with C6ST-1 cDNA totally abolished production of the active C6ST-1. A nearly complete removal of N-glycans of the recombinant C6ST-1 by peptide N-glycosidase F increased the C6ST activity but decreased the KSST activity. Among six potential N-glycosylation sites, deletion of the fourth or sixth site from the amino terminus inhibited production of the active C6ST-1, whereas deletion of the fifth site resulted in a marked loss of the KSST activity. Wild-type recombinant C6ST-1 showed a typical Golgi localization, whereas M-4 recombinant C6ST-1, in which the fourth N-glycosylation site was deleted, colocalized with calnexin, an endoplasmic reticulum-resident protein. Unlike wildtype recombinant C6ST-1, M-4 recombinant C6ST-1 showed a weak affinity toward wheat germ agglutinin and was converted completely to the nonglycosylated form by endoglycosidase H. These observations suggest that N-glycan attached to the fourth N-glycosylation site may function in the proper processing of N-glycans required for the Golgi localization, thereby causing the production of the active C6ST-1, and that N-glycan attached to the fifth N-glycosylation site may contribute to the KSST activity of C6ST-1.

Robust protein quantitation in chromatographic fractions using MALDI‐MS of tryptic peptides

A method is introduced to evaluate protein concentrations using the height sum of all MALDI-MS peaks that unambiguously match theoretic tryptic peptide masses of the protein sought after. The method uses native chromatographic protein fractionation prior to digestion but does not require any depletion, labeling, derivatization, or preparation of a compound similar to the analyte. All peak heights of tryptic peptides are normalized with the peak height of a unique standard peptide added to the MALDI-MS samples. The sum of normalized peak heights, S(n), or the normalized mean peak height, M(n), reflects the concentration of the respective protein. For fractions containing various proteins, S(n) and M(n) can be used to compare concentrations of a protein between different fractions. For fractions with one predominating protein, they can be used to estimate concentration ratios between fractions, or to quantify the fractional protein concentration after calibration with pure protein solutions. Initial native fractionation retains the possibility to apply all conventional analytic procedures. Moreover, it renders the method relatively robust to MS mass accuracy. The method was validated with albumin, transferrin, alpha1-antitrypsin, and immunoglobulin G within highly complex chromatographic fractions of pathological and normal sera, which contained the respective intact native protein in dominating as well as minor concentrations. The correlation found between S(n) and the protein concentration as determined with ELISA showed that the method can be applied to select markers for distinguishing between normal and pathological serum samples.

Use of polysaccharides to control protein adsorption to the air–water interface

In order to understand foaming behaviour of mixed protein/anionic polysaccharide solutions, we investigated the effect of β-lactoglobulin/pectin interaction in the bulk on β-lactoglobulin adsorption to the air–water interface. Adsorption kinetics were evaluated by following surface pressure development in time of several pure protein solutions and of mixed protein/polysaccharide solutions using an automated drop tensiometer (ADT). It was found that complexation of proteins with polysaccharides can slow down the kinetics of surface pressure development by at least a factor 100, and greatly diminish foam formation. In contrast, a five times acceleration in the increase of surface pressure was observed in other cases. We propose a mechanism for protein adsorption from mixed protein/polysaccharide solutions. Effects of ionic strength, pH and mixing ratio on this mechanism were studied for mixtures of β-lactoglobulin and low methoxyl pectin, whereas other proteins and anionic polysaccharides were used to explore the role of protein and polysaccharide charge density and distribution. Whereas the possibilities to change system parameters like ionic strength or pH are limited in food related systems, selecting a suitable combination of protein and polysaccharide offers a broad opportunity to control protein adsorption kinetics and with that foam formation.

Structure and interaction in protein solutions as studied by small-angle neutron scattering

Small-angle neutron scattering (SANS) measurements have been performed to compare the effect of the salts KF, KCl, and KBr on crystallization in aqueous solution of lysozyme protein. It is found that the propensity of the salt to crystallize protein follows the Hoffmeister series (KF<KCl<KBr) with marked differences in the effect of these salts. In pure protein solution, lysozyme macromolecules are prolate ellipsoidal with semimajor and semiminor axes as 22 and 13.5 A, respectively. SANS also gives that the effective (structural+counterion) charge(Z) on the protein as obtained by taking into account screened Coulomb interaction between the protein macromolecules is found to be much smaller than the structural charge. There is decrease in Z suggesting the higher counterion condensation on protein with the increase in the concentration. The counterion condensation seems to be responsible for the differences in the effect of different salts. It is also found that with the addition of salts, lysozyme macromolecules convert to dimers, and for the same salt concentration the comparative effect of different salts follows the Hoffmeister series. Time evolved measurements prior to and after the crystallization show that the protein solution mostly consists of monomers and dimers. Interestingly, higher-mers are not observed in these measurements as perhaps they are formed in very small numbers towards the process that leads to the crystallization. The time dependent data have been used to obtain the fraction of crystallization as a function of time.

Crystallizing proteins on the basis of their precipitation diagram determined using a microfluidic formulator

Crystallization of proteins from a purified protein solution remains a bottleneck in the structure determination pipeline. In this paper the crystallization problem is addressed using a microfluidic device capable of determining detailed protein precipitation diagrams using less than 10 microL of protein sample. Based on the experimentally determined protein phase behavior, a crystallization screen can be designed to accommodate the physical chemistry of the particular protein target. Such a tailor-made crystallization screen has a high probability of yielding crystallization hits. The approach is applied to two different proteins: the calcium pump (SERCA), an eukaryotic integral membrane protein, and UMP kinase, a prokaryotic soluble kinase. Protein phase behavior is mapped for both proteins and tailor-made crystallization screens are designed for the two proteins resulting in about 50% crystallization probability per experiment. This illustrates the power of using microfluidic devices for detailed characterization of protein phase behavior prior to crystallization trials.

Increased Sensitivity of Amino-Arm Truncated βA3-Crystallin to UV-Light–Induced Photoaggregation

Exposure to UV-B light (wavelength, 290-320 nm) is a well-documented risk factor for age-related cataracts. As the lens ages, beta-crystallins tend to undergo proteolytic cleavage of their terminal extensions. To delineate the effects of loss of terminal arms on beta-crystallin function, the sensitivity of purified recombinant wild-type (rbetaA3) to UV-irradiation induced aggregation was compared with that of betaA3-crystallin missing the N-terminal extension (rbetaA3tr). Proteins were expressed in baculovirus-infected Sf9 cells and purified by chromatography. Purified protein solutions (pH 7.4) were reduced by using Tris (2-carboxyethyl) phosphine HCl and irradiated with a 308-nm excimer laser at physiologically relevant UV doses and wavelengths (308 nm), and light-scattering (633 nm) was measured. Irradiated crystallins were analyzed by matrix-assisted desorption ionization (MALDI) and tandem liquid chromatography/mass spectrometry (LC-MS/MS). UV-irradiation of both rbetaA3 and rbetaA3tr resulted in major loss of soluble protein, as shown by absorption at 280 nm, size-exclusion chromatography (SEC) and SDS-PAGE, with concomitant formation of insoluble aggregates producing light-scattering. Compared with wild-type rbetaA3, rbetaA3tr showed a significant tendency to begin scattering light at lower UV dose and had a higher aggregation rate with increasing UV exposure. Changes in irradiated crystallins include aggregation and cross-linking, photolysis, and oxidation of methionine and tryptophan residues. Loss of beta-crystallin terminal arms appears to increase their tendency to aggregate in response to UV irradiation, suggesting that this loss in the maturing lens may increase susceptibility to age-related cataract.

Characterization of a New Pantothenate Kinase Isoform from Helicobacter pylori

Pantothenate kinase (PanK) catalyzes the first step in the biosynthesis of the essential and ubiquitous cofactor coenzyme A (CoA) in all organisms. Two well characterized isoforms of the enzyme are known: a prokaryotic PanK that predominates in eubacteria and a eukaryotic isoform that has primarily been characterized from mammalian and plant sources. Curiously, the genomes of certain pathogenic bacteria, including Helicobacter pylori and Pseudomonas aeruginosa, do not contain a PanK similar to either isoform, although these organisms possess all the other biosynthetic machinery required for CoA production. In this study we cloned, overexpressed and characterized an enzyme from Bacillus subtilis and its homologue from H. pylori and show that they catalyze the ATP-dependent phosphorylation of pantothenate. These enzymes do not share sequence homology with any known PanK, and unlike the bacterial and eukaryotic PanK isoforms their activity is not regulated by either CoA or acetyl-CoA. They also do not accept the pantothenic acid antimetabolite N-pentylpantothenamide as a substrate or are inhibited by it. Taken together, these results point to the identification of a third distinct isoform of PanK that accounts for the only known activity of the enzyme in pathogens such as H. pylori and P. aeruginosa.

Efficient Immobilization of Enzymes on Microchannel Surface Through His-Tag and Application for Microreactor

We developed a simple immobilisation method for His-tagged enzymes on a microchannel surface. It facilitates immobilisation of protein molecule on microchannel surface through Ni-complex, using crude or purified protein solutions. By this method, we could immobilize proteins on microcapillary constantly. This method might be useful for further development of microreactor with reversibly immobilized enzymes.