Zeolites as new chromatographic carriers for proteins--easy recovery of proteins adsorbed on zeolites by polyethylene glycol

Zeolites are able to adsorb proteins on their surface and might be suitable as a new type of chromatographic carrier material for proteins and for their conjugates (Matsui et al., Chem. Eur. J. 7 (2001) 1555-1560). Interestingly, maximum adsorption was observed at the isoelectric point (pI) of each protein. The current study was performed to investigate the desorption of proteins from the zeolites at pI. Proteins adsorbed to zeolites could be desorbed at pI by polyethylene glycol (PEG), but not by conventional eluents. The eluted proteins still retained their activities. The zeolite Na-BEA was an especially good composite for desorption by PEG. Using this method for the adsorption and desorption of proteins at pI, we succeeded in separating various proteins. The application of zeolites to biochemistry and biotechnology is also discussed.     

Affinities of various nucleases to DNA-Sepharose under non-digestive conditions: survey for productive affinity chromatography

1. It has been reported that DNase I can be highly purified from pancreas extract by affinity chromatography on a dDNA-Sepharose column under non-digestive conditions. In the present study, the adsorption-elution of other nucleases on the column under non-digestive conditions was studied. 2. All the seven kinds of nucleases tested were adsorbed when applied on a dDNA-Sepharose column under conditions which did not allow the enzymes to hydrolyze the DNA. The non-digestive conditions were as follows. i) For DNase II (pI=10.2), pH 3.0 in the presence of 50 mM sodium sulfate (inhibitor), ii) for micrococcal nuclease (pI=9.6), pH 4.0 in the absence of Ca2+ (activator), iii) for restriction endonucleases Eco RI (pI=5+1), Hind III (pI=5+1), and Bam HI (pI=5+1), pH 4.0 in the presence of 20% glycerol and 0.1% Neopeptone (stabilizers), and iv) for nucleases S1 (pI=5+1) and nuclease P1 (pI=4.5), pH 7.0. At the respective pH's, the enzymes other than nucleases S1 and P1 were cationic so as to exhibit electrostatic attraction to the anionic dDNA-Sepharose. Although S1 and P1 were anionic, they still adsorbed to the column. 3. All the adsorbed nucleases described above were eluted by a concentration gradient of KCl without changing pH. The ionic strengths required for elution were 0.19 for DNase II, 0.53 for micrococcal nuclease, 0.73 for Eco RI, 0.72 for Hind III, 0.37 for Bam HI, 0.17 for P1, and 0.13 for S1. The fact that the ionic strength required for the elution of DNase I (pI=5.0) was 0.39 at pH 4.0 indicates that the former five enzymes except DNase II can be chromatographed with almost the same or higher efficiency than DNase I, because the proteins adsorbed with no-specific affinity could be mostly eluted at lower ionic strength. On the other hand, the fact that nucleases P1 and S1 were adsorbed in spite of electrostatic repulsion suggests that these two enzymes can also be effectively chromatographed, especially when other cationic proteins are previously removed by an appropriate method such as adsorption to a typical cation exchanger.     

Size-exclusion chromatography of biological samples which contain extremely alkaline proteins

An improved size-exclusion chromatography (SEC) was developed to isolate extremely basic (alkaline) proteins, such as trypsin (pI=10.5), lysozyme (pI=11), and histone (pI=10.8). Develosil 300 Diol-5 (300 x 8 mm I.D., 30 nm average pore diameter) column was used with an eluent of 0.1 M sodium phosphate, 1.5 M sodium chloride, glycerol (40%, v/v), 2-propanol (10%, v/v), and Brij-58 (1%, v/v). Under these conditions, the final apparent pH becomes to 4.0, and pH adjustment is not necessary. Column temperature and flow rate were 15 degrees C and 0.2 ml/min, respectively. This elution system is stable and reliable, and applications onto human pancreatic juice, human bile, and tissue homogenates were successfully achieved. Since this system is convenient for protein analysis, it is expected to be generally applicable to clinical and biochemical research for identifying protein components in combination with microsequencing.     

Comparison of different blocking agents and nitrocelluloses in the solid phase detection of proteins by labelled antisera and protein A

Five different brands of nitrocellulose (NC), each of pore size 0.45 micron and without adsorbed antigen, bound different amounts of two labelled antisera and labelled protein A. Experiments with some non-ionic surface active agents and proteins showed that milk powder and bovine serum albumin were the most effective agents for blocking non-specific binding of labelled protein to NC. With some of the NCs, Nonidet P-40 (NP-40) and Tween 20 were almost as effective as milk powder. The protein-binding capacity of unblocked NC and the level of protein binding after blocking were found to be inversely proportional to the pore size of the NC. A comparison of blocking agents in an immunoassay with pollen proteins adsorbed to NC discs revealed that the highest specific uptakes of antiserum occurred with NP-40 and Tween and not with any of the protein blocking agents such as milk powder. Hence, for the detection of proteins using NC-based assays (but not necessarily following electroblotting), the best choices would appear to be: NC of pore size 0.45 micron; a brand of NC that provides a suitable balance between protein binding capacity and non-specific uptake of protein after blocking; a non-ionic detergent such as NP-40 or Tween 20.     

A pI-based protein fractionation method using solid-state buffers

The analysis of very complex proteomes is dependent on efficient fractionation methods with low level of carry over from fraction to fraction. Among various possibilities the separation by ranges of isoelectric points for further analysis appears as attractive, but current methods involving an electrically driven migration in the presence of ampholyte carriers are not exempt of technical complications. In the present work a new separation concept is described involving the use of so-called solid-state buffers, in association with ion exchangers, to separate protein categories of different pI ranges with a low level of protein overlapping. Resin blends packed in separated columns are used under a cascade configuration of increasing or decreasing pH and, once proteins of different pI are adsorbed by individual resin blends, the columns are dissociated. From each column protein mixtures corresponding to a given pI range are collected by competitive desorption with salts so as to be ready for proteomic analysis. The process is rapid and does not involve electrical fields nor addition of carrier ampholyte material. The presence of potassium chloride during the separation prevents protein precipitation at the vicinicity of their isoelectric points. The fractions thus obtained can be used for two dimensional electrophoresis and mass spectrometry analysis after the removal of salts.     

Protein folding liquid chromatography and its recent developments

The ultimate goal of proteomics is to identify biologically active proteins and to produce them using biotechnology tools such as bacterial hosts. However, proteins produced by Escherichia coli must be refolded to their native state. Protein folding liquid chromatography (PFLC) is a new method developed in recent years, and it is widely used in molecular biology and biotechnology. In this paper, the new method, PFLC is introduced and its recent development is reviewed. In addition the paper includes definitions, advantages, principles, applications for both laboratory and large scales, apparatus, and effecting factors of PFLC. In addition, the role of this method in the future is examined.     

Novel thymine-functionalized polystyrenes for applications in biotechnology. 2. Adsorption of model proteins

This paper investigates the adsorption of bovine serum albumin (BSA) and bovine hemoglobin (BHb) model proteins onto novel thymine-functionalized polystyrene (PS-VBT) microspheres, in comparison with polystyrene (PS) microspheres. Maximum adsorption was obtained for both proteins near their corresponding isoelectric points (pI at pH = 4.7 for BSA and 7.1 for BHb). FTIR and adsorption isotherm analysis demonstrated that, although both proteins were physisorbed onto PS through nonspecific hydrophobic interactions, adsorption onto the functionalized copolymers occurred by both physisorption and chemisorption via hydrogen bonding. FTIR analysis also indicated conformational changes in the secondary structure of BSA and BHb adsorbed onto PS, whereas little or no conformation change was seen in the case of adsorption onto PS-VBT. Atomic force microscopy (AFM), consistent with the isotherm results, also demonstrated monolayer adsorption for both proteins. AFM images of BSA adsorbed onto copolymers with 20 mol % surface VBT loading showed exclusively end-on orientation. Adsorption onto copolymers with lower functionality showed mixed end-on and side-on orientation modes of BSA, and only the side-on orientation was observed on PS. The AFM results agreed well with theoretically calculated and experimentally obtained adsorption capacities. AFM together with calculated and observed adsorption capacity data for BHb indicated that this protein might be highly compressed on the copolymer surface. Adsorption from a binary mixture of BSA and BHb onto PS-VBT showed good separation at pH=7.0; approximately 90% of the adsorbed protein was BHb. The novel copolymers have potential applications in biotechnology.     

Reversible enzyme immobilization via a very strong and nondistorting ionic adsorption on support-polyethylenimine composites

New tailor-made anionic exchange resins have been prepared, based on films of large polyethylenimine polymers (e.g., MW 25,000) completely coating, via covalent immobilization, the surface of different porous supports (agarose, silica, polymeric resins). Most proteins contained in crude extracts from different sources have been very strongly adsorbed on them. Ionic exchange properties of such composites strongly depend on the size of polyethylenimine polymers as well as on the exact conditions of the covalent coating of the solids with the polymer. On the contrary, similar coating protocols yield similar matrices by using different porous supports as starting material. For example, 77% of all proteins contained in crude extracts from Escherichia coli were adsorbed, at low ionic strength, on the best matrices, and less than 15% of the adsorbed proteins were eluted from the support in the presence of 0.3 M NaCl. Under these conditions, 100% of the adsorbed proteins were eluted from conventional DEAE supports. Such polyethylenimine-support composites were also very suitable to perform very strong and nondistorting reversible immobilization of industrial enzymes. For example, lipase from Candida rugosa (CRL), beta-galactosidase from Aspergillus oryzae and D-amino acid oxidase (DAAO) from Rhodotorula gracilis, were adsorbed on such matrices in a few minutes at pH 7.0 and 4 degrees C. Immobilized enzymes preserved 100% of catalytic activity and remained fully immobilized in 0.2 M NaCl. In addition to that, CRL and DAAO were highly stabilized upon immobilization. Stabilization of DAAO, a dimeric enzyme, seems to be due to the involvement of both enzyme subunits in the ionic adsorption.     

包涵体蛋白的层析复性技术研究进展

包涵体蛋白的复性是生物工程下游技术中的一个重要难题。层析法用于蛋白质复性是一种较新的、适用于大多数蛋白的方法。其原理是将层析技术应用于蛋白质复性和纯化,使变性蛋白质在层析柱上重折叠为正确的空间构象,在洗脱的同时实现部分纯化。本文详细介绍了蛋白质在5种层析柱上的复性方法、原理、应用及研究的新进展,为层析法对蛋白质复性的进一步应用提供依据。     

Two step procedure for purification of enzymatically active prostate-specific antigen from seminal plasma

The role of prostate-specific antigen (PSA) during the onset of prostate cancer and subsequent tumor growth and metastasis is not well understood. We have developed a simple two step procedure, based on principles of hydrophobic charge-induction chromatography and molecular size chromatography to provide pure free-PSA (f-PSA) preparation that is free from all other known PSA complexes as well as human kallikrein 2 (hK2). The overall recovery of f-PSA is 72%. The isolated f-PSA consists of three known isoforms that corresponds to pI of 6.2, 6.4 and 7.2. f-PSA is enzymatically active and its enzymatic activity can be effectively neutralized by a serine protease inhibitor.     

Protein chromatography in neat organic solvents

Pure formamide and ethylene glycol are used instead of water as processing media for protein chromatography. A number of common proteins are freely soluble in these solvents and most do not undergo irrersible inactivation in them. Batch adsorption studies reveal that proteins readily adsorbed to various ion-exchangers in formamide and ethyline glycol and subsequently can be completely desorbed by adding inorganic salts (LiCl and NH(4)NO(3)) to the solvents. The idea of protein separations in formamide and ethylene glycol is illustrated by column chromatography and preparative separation of mixtures of (i) oxidized A and B chains of insulin and (ii) lysozyme and ribonuclease on the anion-exchanger triethylaminoethycellulose and the cation-exchanger phosphocellulose, respectively.     

Amphoteric, buffering chromatographic beads for proteome prefractionation. I: theoretical model

The possibility is reported here of fractionating proteins on amphoteric, buffering resins via ion-exchange chromatography. A given protein's adsorption to a particular amphoteric buffering resin is characterized by a bell-shaped curve in which the maximum protein binding capacity is observed at an optimum pH value lying approximately midway between the isoelectric point values (pI) of the resin and the protein. On either side of this maximum the protein binding capacity declines steadily, reaching zero at the pI of either the protein or exchanger. For instance, on beads of pI equal to 8, four proteins, two acidic (bovine albumin and ovalbumin) and two basic (cytochrome c and lysozyme), exhibit binding curves reaching zero values for the whole set when the exchanger is conditioned at pH 8.0. Away from the pI, and on both sides of the pH scale, the bell-shaped adsorption curves reach a maximum, for each protein, at a pH located at the midpoint between the pI values of each protein and that of the exchanger, and decline steadily to reach zero at the pI value of each protein species. Separation of model proteins using different amphoteric buffering resins of various pI was possible at different pH values according to both the pI of the proteins and of the exchangers. It was also demonstrated, using surface enhanced laser desorption/ionization mass spectrometry and two dimensional electrophoretic mapping, that separation of an Escherichia coli cell lysate on columns packed with amphoteric buffering resins of different pI and titrated to a particular pH value, delivered two distinctly different fractions, i.e. characteristically composed of, on the one hand, proteins having a pI below the buffer pH (the 'adsorbed' fraction), and on the other, of alkaline proteins possessing a pI above the pH of the buffer (the 'unadsorbed' fraction). This approach represents an attractive addition and/or alternative to the armory of protein pre-fractionation techniques currently employed in proteomics.     

Delivery of bone morphogenetic proteins for orthopedic tissue regeneration

Carriers for bone morphogenetic proteins (BMPs) are used to increase retention of these factors at orthopedic treatment sites for a sufficient period of time to allow regenerative tissue forming cells to migrate to the area of injury and to proliferate and differentiate. Carriers can also serve as a matrix for cell infiltration while maintaining the volume in which repair tissue can form. Carriers have to be biocompatible and are often required to be bioresorbable. Carriers also have to be easily, and cost-effectively, manufactured for large-scale production, conveniently sterilized and have appropriate storage requirements and stability. All of these processes have to be approvable by regulatory agencies. The four major categories of BMP carrier materials include natural polymers, inorganic materials, synthetic polymers, composites of these materials. Autograft or allograft carriers have also used. Carrier configurations range from simple depot delivery systems to more complex systems mimicking the extracellular matrix structure and function. Bone regenerative carriers include depot delivery systems for fracture repair, three-dimensional polymer or ceramic composites for segmental repairs and spine fusion and metal or metal/ceramic composites for augmenting implant integration. Tendon/ligament regenerative carriers range from depot delivery systems to three-dimensional carriers that are either randomly oriented or linearly oriented to improve regenerative tissue alignment. Cartilage regenerative systems generally require three-dimensional matrices and often incorporate cells in addition to factors to augment the repair. Alternative BMP delivery systems include viral vectors, genetically altered cells, conjugated factors and small molecules.     

Steroid receptors analysis in human mammary tumors by isoelectric focusing in agarose

A high resolution and quantitative method for isoelectric focusing has been developed to separate the isoforms of estrogen and progesterone receptors in human mammary tumor cytosols stabilized by sodium molybdate. Agarose gels (0.5%) were used. Six samples can be analyzed on one gel in about 2 h, and 35-microliters samples are sufficient to determine the estrogen receptor isoform pattern. The constant yields and the reproducibility of data allow a quantitative analysis of these receptors. Four estrogen receptor isoforms have been observed (pI 4.7, 5.5, 6, and 6.5), isoforms with pI 4.7 and 6.5 being present in all tumors. After incubation at 28 degrees C in high ionic strength, the comparison of isoelectric focusing and high-performance size exclusion chromatography patterns of estrogen receptor confirms the oligomeric structure of the pI 4.7 isoform and suggests a monomeric structure for the pI 6.5 isoform. Under the same conditions of analysis, only one progesterone receptor isoform has been detected with pI 4.7.     

Mapping of surface glycoproteins of Trypanosoma cruzi by two-dimensional electrophoresis. A correlation with the cell invasion capacity

The cell-surface iodinatable proteins of Trypanosoma cruzi have been analyzed by two-dimensional polyacrylamide gel electrophoresis under equilibrium conditions. Antigenic polypeptides were characterized after immunoprecipitation and glycoproteins were identified by means of lectin-affinity chromatography. Two glycoproteins, with affinity for concanavalin A, were found to be common to both infective (trypomastigote) and non-infective (epimastigote) forms: protein 1 (90 kDa, pI 5.5-6.5) and protein 2 (80 kDa, pI 5.3-6.3). In epimastigotes a specific concanavalin-A-binding surface glycoprotein (70 kDa, pI 5.5) was identified. Trypomastigote forms, on the other hand, presented several specific iodinatable surface components: glycoproteins 3(85 kDa, pI 5.5), 4 (85 kDa, pI 5.0), 6 (100 kDa, pI 6.5), 7 (120 kDa, pI 6.3), 8 (68 kDa, pI 6.7) and several minor high-molecular-mass acid proteins, all containing glucose and/or mannose, and glycoprotein 5 (85 kDa, pI 6.3-7.5), containing N-acetyl-D-glucosamine (Tc-85). Proteins 1, 2 and 5 were the only ones which gave clear evidence of charge heterogeneity. Most of the surface proteins of trypomastigote forms, the exception being proteins 3, 4 and 8, were removed by treatment with trypsin. This proteolytic treatment results in 90% inhibition of the in vitro vertebrate-cell-invasion capacity of the parasites. Upon reincubation in culture medium for 4 h, the trypsin-removed glycoproteins are again detected, an observation that correlates well with the recovery of the cell-penetration capacity observed in the same period.     

Analysis of rat serum apolipoproteins by isoelectric focusing. II. Studies on the low molecular weight subunits.

The low molecular weight proteins of rat apo HDL and apo VLDL have been isolated and analyzed by the technique of isoelectric focusing. Sephadex fractions from apo HDL (HS-3) and apo VLDL (VS-3) that contain these proteins reveal three major bands with apparent isoelectric points of pH 4.50, 4.67, and 4.74, as well as three minor bands at pH 4.43, 4.57, and 4.61. In addition, apo HDL has a major band at pI of 4.83. DEAE-Cellulose chromatography was used to prepare purified fractions of these components that were characterized by N-terminal analyses and molecular weight determinantions by SDS gel electrophoresis. The major low molecular weight components of apo HDL were focused on a slab gel and the bands were identified as A-II (pI 4.83), C-II (pI 4.74), C-III-0 (pI 4.67), and C-III-3 (pI 4.50). Neuraminidase treatment of apo HDL, followed by isoelectric focusing, suggested that the other bands, which have not previously been reported, may be additional forms of the C-III protein, differing only in their content of sialic acid.     

Aptamers as functional nucleic acids:<Emphasis Type="Italic">In vitro</Emphasis> selection and biotechnological applications

Aptamers are functional nucleic acids that can specially bind to proteins, peptides, amino acids, nucleotides, drugs, vitamins and other organic and inorganic compounds. The aptamers are identified from random DNA or RNA libraries by a SELEX (systematic evolution of ligands by exponential amplification) process. As aptamers have the advantage, and potential ability to be released from the limitations of antibodies, they are attractive to a wide range of therapeutic and diagnostic applications. Aptamers, with a high-affinity and specificity, could fulfil molecular the recognition needs of various fields in biotechnology. In this work, we reviewed some aptamer selection techniques, properties, medical applications of their molecules and their biotechnological applications, such as ELONA (enzyme linked oligonucleotide assay), flow cytometry, biosensors, electrophoresis, chromatography and microarrays.     

Purification of Escherichia coli 30S ribosomal proteins by high performance liquid chromatography

High performance liquid chromatography was applied to the separation of proteins derived from the Escherichia coli 30S ribosomal subunit. Several methods of separating this protein mixture has been tested: size-exclusion chromatography on hydrophilic phases; ion exchange and reversed phase chromatography (on C2 to C18 hydrocarbon-bonded supports). Various elution systems were examined in order to obtain pure proteins suitable for micro-sequence analysis. The resolution and yields of the proteins varied considerably, depending on the type of support and gradient system used. The best results were achieved with uniformly globular-shaped supports of large pore size, and by combining high performance size exclusion with rechromatography on reversed phase columns. Purification conditions for the individual proteins are listed. The methods employed avoid any precipitation step and allow easy identification of the proteins by one or two-dimensional gel electrophoresis, amino-acid analysis or direct manual or automatic micro-sequencing. Since the isolation time is much reduced compared with conventional purification procedures, the proteins obtained by the techniques described here are well suited for topographical and immunological studies or reconstitution assays. Ribosomal proteins of other organisms can be separated under similar conditions.     

Proteome analysis by three-dimensional protein separation: turnover of cytosolic proteins in hepatocytes

We performed a three-dimensional separation of pulse-chase dual-labelled rat liver cytosolic proteins using hydrophobic interaction chromatography, isoelectric focusing, and SDS gel electrophoresis. Due to very different expression rates but similar size and pI of rat liver cytosolic proteins, we demonstrate the impossibility of successful two-dimensional separations of such complex protein mixtures. A pre-fractionation of proteins by hydrophobic interaction chromatography is therefore recommended prior to two-dimensional gel electrophoresis. Our studies confirmed the correlation between protein turnover rates and surface hydrophobicity.     

Mitochondrial carriers function as monomers

Mitochondrial carriers link biochemical pathways in the mitochondrial matrix and cytosol by transporting metabolites, inorganic ions, nucleotides and cofactors across the mitochondrial inner membrane. Uncoupling proteins that dissipate the proton electrochemical gradient also belong to this protein family. For almost 35 years the general consensus has been that mitochondrial carriers are dimeric in structure and function. This view was based on data from inhibitor binding studies, small-angle neutron scattering, electron microscopy, differential tagging/affinity chromatography, size-exclusion chromatography, analytical ultracentrifugation, native gel electrophoresis, cross-linking experiments, tandem-fusions, negative dominance studies and mutagenesis. However, the structural folds of the ADP/ATP carriers were found to be monomeric, lacking obvious dimerisation interfaces. Subsequently, the yeast ADP/ATP carrier was demonstrated to function as a monomer. Here, we revisit the data that have been published in support of a dimeric state of mitochondrial carriers. Our analysis shows that when critical factors are taken into account, the monomer is the only plausible functional form of mitochondrial carriers. We propose a transport model based on the monomer, in which access to a single substrate binding site is controlled by two flanking salt bridge networks, explaining uniport and strict exchange of substrates.