Polybuffer Exchanger Research Articles

Purification of pituitary autoantigen by column liquid chromatography and chromatofocusing.

IntroductionPituitary autoantibodies can be determined both in patients with pituitary disease as well as patients with autoimmune endocrine diseases. The purpose of the study was to isolate and purify pituitary autoantigen using sera of patients and the microsomal fraction of the pituitary.Material and methodsTo isolate a pituitary autoantigen, patient sera were used, which showed a strong immune response to pituitary antigens. Pituitary microsomal fractions were prepared from pituitary tissue homogenates. In the study, sera of patients with pituitary disease, Addison and Graves’ disease were used. The initial stages were carried out by affinity chromatography on CN -Br sepharose column whereas purification was continued by column liquid chromatography on AcA54 Ultrogel. Chromatofocusing was performed by Polybuffer exchanger PBE 94.Results 125I-labeled pituitary antigens after isolation appeared in column chromatography in three peaks. The first peak contained 50-70 kDa proteins, the second peak – 17 to 22 kDa proteins and the third peak contains 125-iodides. Three fractions obtained from filtration on Ultrogel were separated in a polyacrylamide gel. In the first peak two bands 67 and 55 kDa appeared. The second peak contained low molecular weight substances, and the third peak contained 125I. The first peak from Ultrogel was isolated by chromatofocusing – the first peak with pH 5.9 and the second one with pH 4.9.ConclusionsIsolation and purification of pituitary autoantigen with the use of column liquid chromatography and chromatofocusing resulted in obtainment of two antigenic proteins of specific gravity of 67 and 55 kDa.

Purification and characterisation of two acidic peroxidase isoforms from the sheaths of bamboo shoots

SummaryThree isoforms of peroxidase (POD) were isolated from the sheaths of bamboo shoots by chromatofocusing on Polybuffer exchanger PBE 94. POD‐A was partially purified, and POD‐B and POD‐C were purified and characterised. POD‐A was a basic peroxidase, whereas POD‐B and POD‐C were acidic peroxidases with different isoelectric points. Using o‐phenylenediamine (OPD) as a hydrogen‐donor, the optimum temperatures for function of POD‐B and POD‐C were 55 and 45–55 °C, respectively, while both had the same optimum pH of 4.5. Both isoforms were stable between 30 and 60 °C and between pH 4.5 and 10. The activities of the POD isoforms towards hydrogen‐donors were both pH and concentration dependent. Under optimal conditions, POD‐B and POD‐C catalysed the oxidation of catechol, pyrogallol, and OPD at higher rates than guaiacol, o‐dianisidine and 2, 2’‐azino‐bis‐ (3‐ethylbenzothiazoline‐6‐sulphonic acid). Both isoforms were almost completely inhibited by chemical modification reagent diethyl pyrocarbonate (4.5 mM).

Preparation of the Gel and Column for Chromatofocusing: Figure 1.

INTRODUCTIONThe appropriate stationary phase for chromatofocusing depends very much on the intended application. In general, in order to form a linear pH gradient (which is critical for good separation), the stationary phase must provide uniform buffering capacity over the pH separation range of interest. Thus, the functional groups on the matrix must be able to provide high buffering capacity over a broad pH range. Examples of such functional groups are polyethyleneimine (PEI) and a mixture of tertiary and quaternary amines. The most commonly used stationary phases for chromatofocusing are polymer-based stationary phases such as Mono P or polybuffer exchanger (PBE). In this protocol, a column is readied and packed with PBE matrix. The matrix is equilibrated with the buffer that will be used at the start of the separation, and the quality of the packing is assessed using cytochrome c.

Chromatofocusing

INTRODUCTIONChromatofocusing separates proteins on the basis of differences in their isoelectric point (pI). The stationary phase (matrix) is usually a weak anion exchanger in which the functional groups are amines, for example, a polybuffer exchanger (PBE 118 and PBE 94) or Mono P column. The eluent is a buffer containing a large number of buffering species that together give a uniform buffering capacity over a broad pH range.

Preparation of highly purified F-actin-depolymerizing factor of human serum

F-actin depolymerizing factor (ADF) of human serum was purified 250-400-fold to more than 98% purity with high reproducibility. The purification included (1) 30-50% (NH4)2SO4 precipitation, (2) ion-exchange chromatography on DEAE-Sepharose, (3) chromatofocusing on Polybuffer exchanger 94 and (4) affinity chromatography on ConA-Sepharose. The recovery of ADF was estimated to be 20-30% whereas the ADF activity yield was 5-17%. The lower activity yield was thought to be due-partly to proteolysis and partly to destabilization of highly purified ADF.

β-glucosidase from Sclerotinia sclerotiorum: a new and efficient purification procedure and use as a suitable marker in immuno-enzymatic assay

The β-glucosidase enzyme β-glu2 isolated from Sclerotinia sclerotiorum was purified and used as tracer in enzyme linked immunosorbent assay. A novel purification procedure of the protein was developed that consists of ammonium sulphate precipitation, gel filtration on Sephacryl S-200-HR column, ion exchange chromatography on DEAE-Toyopearl and polybuffer exchanger PBE 94 TM chromatofocusing. The pI value was 4.45. Kmand Vmax values for the enzyme towards p-nitrophenyl-β-D-glucopyranoside were respectively 0.45 mM and 0.2 U/mL. Thermal stability showed that β-glu2 has a half-life of 85 min at 55 °C and of 25 min at 65 °C. β-glu2 was conjugated to goat anti-rabbit antibodies with glutaraldehyde as cross linking agent according to the one-step method. Conjugates were purified by HPLC gel filtration on TSK 2000. Enzymatic and immunological activities of the β-glucosidase conjugate component were tested by the ELISA method.

Catalytic Characteristics of Peroxidase from Wheat Grass

The crude enzyme extract of wheat grass was heated at 60 degrees C for 30 min, followed by ammonium sulfate fractionation and isoelectric chromatofocusing on Polybuffer exchanger (PBE 94) for purification. The purified peroxidase was then characterized for its catalytic characteristics. It was found that AgNO3 at a concentration of 0.25 mM and MnSO4 and EDTA at concentrations of 5 mM significantly inhibited the activity of wheat grass peroxidase. However, KCl, NaCl, CuCl2, CaCl2, ZnCl2, and MgCl2 at concentrations of 5.0 mM and HgCl2 at a concentration of 0.25 mM enhanced enzyme activity. Chemical modification significantly influenced the activity of wheat grass peroxidase. Particularly, N-bromosuccinimide (5 mM) inhibited 16% of the enzyme activity, whereas N-acetylimidazole (2.5 mM), diethyl pyrocarbonate (2.5 mM), and phenylmethanesulfonyl fluoride (2.5 mM) enhanced by 18-29% of the enzyme activity. Such results implied that tryptophan, histidine, tyrosine, and serine residues are related to enzyme activity. The pH optima for wheat grass peroxidase to catalyze the oxidation of o-phenylenediamine (OPD), catechol, pyrogallol, and guaiacol were 5.0, 4.5, 6.5, and 5.0, respectively. The apparent Km values for OPD, catechol, pyrogallol, and guaiacol were 2.9, 18.2, 2.5, and 3.8 mM, respectively. Under optimal reaction conditions, wheat grass peroxidase catalyzed the oxidation of OPD (an aromatic amine substrate) 3-11 times more rapidly than guaiacol, catechol, and pyrogallol (phenolic substrates containing one to three hydroxy groups in the benzene ring).

Purification of Taenia solium cysticerci superoxide dismutase and myoglobin copurification.

Superoxide dismutase from Taenia solium cysticerci ( Ts SOD) was purified by sequential ion exchange chromatography on quaternary-amino-ethyl-cellulose (QAE) followed by hydrophobic interaction on phenyl sepharose (PS) and chromatofocusing on a polybuffer exchanger 94 (PBE). Ts SOD is a 30 kDa molecular weight dimeric enzyme with 15 kDa monomers. It is partially negative, hydrophilic, with 6.3 isoelectric point and has 2,900 U/mg activity. Bovine erythrocyte SOD antibodies cross react with Ts SOD. This enzyme is 80% inhibited by 10 mM of KCN suggesting that it has a Cu/Zn active site. Furthermore, Ts SOD totally loses its activity at 100 degrees C for 4 min. The first 25 amino acids from the Ts SOD N-terminal are (M K A V X V M R G E E G V K G V V H F T Q A G D A). This sequence is 76% similar to the Schistosoma mansoni Cu/Zn SOD. By chance, myoglobin (Mb) was also found during the purification process. A 16 kDa band was recognized in immunoblotting by horse heart Mb antibodies in QAE, PS and PBE, the last-mentioned being found at pH 7.0. The first 15 amino acids from the amino terminal group (G L S D G E W Q L V L N V W G) in this 16 kDa protein are identical to several other Mbs which have been reported.

Heterologous expression in Pseudomonas aeruginosa and purification of the 9.2-kDa c-type cytochrome subunit of p-cresol methylhydroxylase.

The 9.2-kDa c-type cytochrome subunit (PchC) of the flavocytochrome p-cresol methylhydroxylase from Pseudomonas putida NCIMB 9869 has been overexpressed in recombinant form in Pseudomonas aeruginosa PAO1-LAC, using the recently developed pUCP-Nde vector. Efforts to produce the cytochrome in Escherichia coli using a pET vector, with or without its signal peptide, were generally unsuccessful, yielding relatively low levels of the protein. In contrast, the mature form of PchC accumulated in the periplasmic space of P. aeruginosa PAO1-LAC to about 1 mg/g wet cell paste. A periplasmic fraction enriched to about 12% (w/w) of total protein with recombinant PchC was isolated from the remainder of the cells by a washing procedure using ethylenediaminetetraacetate in the presence of sucrose. The cytochrome was purified to homogeneity from the periplasmic extract by anion-exchange chromatography on DEAE-Sepharose CL-6B followed by chromatofocusing on PolyBuffer Exchanger 94. Purified PchC was obtained in a yield of about 50% and was shown to be identical to that resolved from the native flavocytochrome isolated from P. putida. This system may prove to be of general use for the production of recombinant c-type cytochromes.

Purification to homogeneity and characterization of a novel Pseudomonas putida chromate reductase.

Cr(VI) (chromate) is a widespread environmental contaminant. Bacterial chromate reductases can convert soluble and toxic chromate to the insoluble and less toxic Cr(III). Bioremediation can therefore be effective in removing chromate from the environment, especially if the bacterial propensity for such removal is enhanced by genetic and biochemical engineering. To clone the chromate reductase-encoding gene, we purified to homogeneity (>600-fold purification) and characterized a novel soluble chromate reductase from Pseudomonas putida, using ammonium sulfate precipitation (55 to 70%), anion-exchange chromatography (DEAE Sepharose CL-6B), chromatofocusing (Polybuffer exchanger 94), and gel filtration (Superose 12 HR 10/30). The enzyme activity was dependent on NADH or NADPH; the temperature and pH optima for chromate reduction were 80 degrees C and 5, respectively; and the K(m) was 374 microM, with a V(max) of 1.72 micromol/min/mg of protein. Sulfate inhibited the enzyme activity noncompetitively. The reductase activity remained virtually unaltered after 30 min of exposure to 50 degrees C; even exposure to higher temperatures did not immediately inactivate the enzyme. X-ray absorption near-edge-structure spectra showed quantitative conversion of chromate to Cr(III) during the enzyme reaction.

307 Characterization of Amylolytic Activities of Tulip Bulb Scales

Amylolytic activities extracted from scales of tulip (Tulipa gesneriana L. cv. Apeldoorn) bulbs stored at 4 °C for 6 weeks under moist conditions were characterized. Anion exchange chromatography of enzyme extract on DEAE-Sephacel revealed three peaks of amylolytic activity. Three enzymes showed different electrophoretic mobilties on nondenaturing polyacrylamide gels. The most abundant amylase activity was purified extensively with phenyl-agarose chromatography, gel filtration on Sephacryl S-200, and chromatofocusing on polybuffer exchanger PBE 94. The purified amylase was determined to be an endoamylase based on substrate specificity and end product analysis. The enzyme had a pH optimum of 6.0 and a temperature optimum of 55 °C when soluble starch was used as the substrate. The apparent Km value for soluble starch was 1.28 mg/ml. The inclusion of 2 mM CaCl2 in the reaction mixture resulted in a 1.4-fold increase in the enzyme activity. The presence of calcium ions also enhanced the thermo-stability of the enzyme at higher temperatures. The enzyme was able to hydrolyze soluble starch, amylose, amylopectin, and beta-limit dextrin, but it had no activity against pullulan, inulin, maltose, or p-nitrophenyl alpha-glucopyranoside. Only maltooligosaccharides, having a degree of polymerization of 7 or more, were hydrolyzed to a significant extent by the enzyme. Exhaustive hydrolysis of soluble starch with the enzyme yielded a mixture of maltose and matlooligosaccharides. This amylase activity was not inhibited by alpha- or beta-cyclodextrin upto a concentration of 10 mM. Maltose at a 50 mM concentration partially inhibited the enzyme activity, whereas glucose had no effect at that concentration.

Purification and Biochemical Properties of Beta-Lactamase fromEscherichia Coli

A β-lactamase was purified to homogeneity from a clinical isolate of Escherichia coli by filtration on Sephadex G-75 and chromatofocusing on a polybuffer exchanger PBE94 column followed by chromatography on phenyl-Sepharose CL-4B. Although the purified enzyme is from a gram-negative bacterial source. it presented several common features of the gram-positive bacterial β-lactamases. After punfication. a single polypeptide with molecular size of 30,000 was found in sodium dodecylsulfate-polyacrylamide gel electrophoresis and enzyme activity that migrated at an isoelectric point of 5.4 was inhibited by clavulanic acid, but not by aztreonam, EDTA and NaCl.

Enrichment and purification of proteins of Haemophilus influenzae by chromatofocusing

Haemophilus influenzae is a bacterium of pharmaceutical interest of which the entire genome has been sequenced. Identification of low-abundance proteins in a two-dimensional map is important for the detection of new drug targets. We applied chromatography on Polybuffer Exchanger (chromatofocusing) in order to fractionate and enrich H. influenzae proteins, possibly low-copy-number gene products, from larger volumes. Two proteins, major ferric iron-binding protein (HI0097) and 5′-nucleotidase (HI0206) were obtained in pure form and hypothetical protein HI0052 was purified to near homogeneity by this single purification step. Four other proteins, aspartate ammonia lyase (HI0534), peptidase D (HI0675), elongation factor Ts (HI0914) and 5-methyltetrahydropteroyltriglutamate methyltransferase (HI1702), were strongly enriched so that chromatography on Polybuffer Exchanger can be used as an initial step for their isolation. Approximately 125 proteins were identified in the fractions collected from the column. Seventy of these were for the first time identified after chromatography on Polybuffer Exchanger. The proteins enriched by the chromatofocusing step include both low-abundance as well as high-copy-number gene products. They do not belong to a single protein class and the majority of them are enzymes with various functions. The results include a list and a two-dimensional map of the proteins enriched by chromatofocusing. They may be useful in the search of drug targets and in the design of purification protocols for the isolation of homologous proteins from related microorganisms.

A cell wall-bound β-Glucosidase from germinated rice: Purification and properties

A cell wall-bound β-glucosidase was purified from germinated rice seeds, and its N-terminal amino acid sequence was determined. The enzyme had a molecularmass of 56 kDa and an isoelectric point of > 10.0. The enzyme showed hydrolytic as well as trans-glycosylation activity towards β-linked oligosaccharide substrates, suggesting that it may be involved in hydrolysis as well as reorganization of oligosaccharides in cell walls during germination. A large portion of β-glucosidase (EC 3.2.1.21) in germinating rice seeds, which appears to be ionically bound to cell walls, can be solubilized with 1 M NaCl. Its activity increased more than eight-fold within five days of germination. It was purified to electrophoretic homogeneity from the extracts of germinated rice seeds by fractionation with (NH 4) 2SO 4 followed by CM-Sepharose, Polybuffer exchanger 118, Concanavalin A-Sepharose and Bio-Gel P-100. The Mr of the purified enzyme, estimated by SDS-PAGE, was 56,000 and the isoelectric point was > 10.0. Its N-terminal amino acid sequence (44 residues) exhibited high homology to those of β-glucosidases from other plants, such as barley and white clover. Its activity was optimal at pH 4.5 and 50°, and it was strongly inhibited by glucono-1,5-lactone. The enzyme showed hydrolytic as well as transglycosylation activity towards (1 → 3)-β- and (1 → 4)-β-linked oligosaccharides with degree of polymerization of 2–4. The results suggest that the β-glucosidase is probably involved not only in hydrolysis but also in modification of oligosaccharides in cell walls of germinating rice seeds.

AP30, a differential protein marker for perilymph and cerebrospinal fluid in middle ear fluid, has been purified and identified as human apolipoprotein D

Using two-dimensional (2-D) gel electrophoresis, human perilymph and cerebrospinal fluid have been shown to be highly enriched for an acidic protein with Mr 30 000, we designated it as AP30. The protein exhibits charge heterogeneity, with at least eight isoforms visible between pI 4.5 to 5.5 on 2-D gels. Purification of the protein was carried out by ammonium sulfate precipitation, polybuffer exchanger column chromatofocusing, and acetone fractional precipitation. The resulting preparation also contains eight spots in the acidic area of 2-D gels, and one broad band located at Mr 30 000 by SDS-PAGE. Digestion of AP30 with neuraminidase causes the isoforms to shift to a more basic position and to consolidate into two primary spots, indicating that AP30 is a variably sialylated glycoprotein. Amino acid analysis of AP30 revealed an amino acid content very similar to that of human apolipoprotein D. Attempts to determine the amino acid sequence demonstrated that the N-terminus is blocked. Edman sequencing of two peptide fragments, generated by cyanogen bromide cleavage of AP30, both revealed sequences having 100% identity to human apolipoprotein D. Western blot analysis of AP30 with the antibody against authentic human apolipoprotein D demonstrated a high degree of cross-reactivity. These studies indicate that AP30 from human perilymph and cerebrospinal fluid is a member of the apolipoprotein D family.

Purification, characterization and NH 2-terminal sequencing of an endo-(1→3,1→4)- β-glucanase from rice ( Oryza sativa L.) bran

A (1→3,1→4)- β-glucanase was purified from rice bran to electrophoretic homogeneity by fractionation with ammonium sulfate followed by ion-exchange chromatography’s on DEAE- and CM-Sepharose, chromatofocusing on Polybuffer exchanger 94 and hydrophobic interaction chromatography on Phenyl-Sepharose. The purified enzyme had a molecular mass of 31 kDa as judged by SDS-PAGE and a basic isoelectric point of 7.9. The enzyme specifically hydrolyzed (1→3,1→4)- β-glucans such as β-glucans of barley and oat, and lichenins, but not CM-cellulose and laminarin. The purified enzyme was most active at pH 4.5 and stable for 10 min at 40°C. When barley (1→3,1→4)- β-glucan was used as the substrate, two oligosaccharides were formed as the major products of hydrolysis after prolonged incubation with the enzyme. No glucose was detected however, indicating that the enzyme may be classified as an endo-(1→3,1→4)- β-glucanase (EC 3.2.1.73). NH 2-terminal amino acid sequencing (40 residues) of rice endo-(1→3,1→4)- β-glucanase revealed that it had a high similarity (83–85%) to that of endo-(1→3,1→4)- β-glucanase from barley and wheat.

Purification and immobilization of dextranase

AbstractAn enzymic characteristic of Novo dextranase was presented. In addition to a high dextranolytic activity (7,200 U/ml), the crude enzyme also contained small amounts of protease, glucoamylase, polygalacturonase, carboxymethylcellulase, laminarinase and chitinase. A highly purified dextranase was then simply separated from a commercial preparation by column chromatographies on DEAE‐Sepharose, CM‐Sepharose, and by chromatofocussing on Polybuffer Exchanger PBE‐94. The enzyme was recovered with an over 200‐fold increase in specific activity and a yield of 84%. The final preparation was homogeneous, as observed during high performance liquid chromatography (HPLC). Size‐exclusion HPLC indicated that dextranase had a molecular mass of 35 kDa and its isoelectric point, established by chromatofocussing, was 4.85. Analysis of the dextran break‐down products indicated that purified dextranase represents an endolytic mode of action, and isomaltose and isomaltotriose were identified as the main reducing sugars of dextran hydrolysis. The enzyme was then covalently coupled to the silanized porous glass beads modified by glutaraldehyde (Carrier I) or carbodiimide (Carrier II). It was shown that immobilization of dextranase gave optimum pH and temperature ranges from 5.4 to 5.7 and from 50°C to 60°C, respectively. The affinity of the enzyme to the substrate decreased by a factor of more than 13 for dextranase immobilized on Carrier I and increased slightly (about 1.4‐times) for the enzyme bound to Carrier II.

Purification of Storage Granule Protein-23: A NOVEL PROTEIN IDENTIFIED BY PHAGE DISPLAY TECHNOLOGY AND INTERACTION WITH TYPE I PLASMINOGEN ACTIVATOR INHIBITOR

Type 1 plasminogen activator inhibitor (PAI-1) is a key regulator of the fibrinolytic cascade that is stored in a rapidly releasable form within platelet alpha-granules. To identify proteins that may participate in the targeting or storage of this potent inhibitor, this report investigates the applicability of utilizing filamentous bacteriophages to display proteins expressed by cells containing a regulated secretory pathway and their enrichment based upon an interaction with PAI-1. For this purpose, RNA was extracted from AtT-20 cells (i.e. a classical model cell system for intracellular protein sorting), reverse transcribed, amplified using polymerase chain reaction primers containing internal restriction sites, and cloned into the phagemid pCOMB3H for expression as fusion constructs with the bacteriophage gene III protein. Escherichia coli was transformed with the phagemids and infected with VCSM13 helper phage, and the resulting AtT-20 cDNA-bacteriophage library was enriched by panning against solid- and solution-phase PAI-1. The enriched cDNA library was subcloned into a prokaryotic expression vector system that replaces the gene III protein with a decapeptide tag for immunologic quantitation. One novel cDNA clone (i.e. A-61), which preferentially recognized solution-phase PAI-1 and reacted positively with antibodies derived from a rabbit immunized with alpha-granules, was subcloned into the prokaryotic expression vector pTrcHis to create a construct containing an N-terminal six-histidine purification tag. This construct was expressed in E. coli, purified by nickel-chelate chromatography followed by preparative SDS-polyacrylamide gel electrophoresis, and utilized for the generation of polyclonal antibodies. Immunoblotting analysis employing antibodies against the purified A-61 construct revealed a 23-kDa protein present in the regulated secretory pathway of AtT-20 cells. The 23-kDa molecule was purified from media conditioned by AtT-20 cells by ion exchange chromatography on DEAE-Sephacel, molecular sieve chromatography on Sephacryl S-100, chromatofocusing on Polybuffer exchanger 94, and affinity chromatography on PAI-1-Sepharose. N-terminal amino acid sequencing of a 16-kDa Lys-C proteolytic fragment of the 23-kDa storage granule protein was employed to confirm its identity with the cDNA sequence of clone A-61. These data indicate that phage display of cDNA libraries fused to the C-terminal region of the gene III protein and their enrichment via an interaction with a target molecule can be utilized to define other proteins present within a particular cellular pathway.

Purification of P26h: a hamster sperm protein.

P26h is a 26 kDa glycoprotein, located on the acrosome cap of hamster spermatozoa, involved in the species specificity of gamete interaction. We have purified this protein from hamster spermatozoa collected from the distal cauda region of the epididymis. Its purification was realized following a three-step procedure: detergent extraction, ion-exchange chromatography, and chromatofocusing. Protein partitioning using Triton X-114 (the first step) showed a ratio of 5:1 between the resulting aqueous and detergent phase. P26h was found almost exclusively in the aqueous phase where it represented about 10-12% of the total protein content. When the desalted aqueous phase was loaded on a carboxymethyl column for cation-exchange chromatography, about 80% of the proteins did not bind to the matrix and were eliminated. P26h was eluted from the column with a linear gradient of salt. At this point, P26h had a rate of purification estimated at 45-55%; three other major proteins of <21, 45, and 63 kDa remained in the sample. These undesired proteins were eliminated by submitting these samples to chromatofocusing using a PBE 94 polybuffer exchanger column. Indeed, P26h was collected almost in the dead volume of the column while the other proteins were eluted much later. Two-dimensional gel electrophoresis and Western blotting were performed to determine the purity of P26h. Only one major spot was detected, confirming the purity of P26h. Usefulness of this purified sperm antigen in the understanding of the physiology of mammalian fertilization is discussed.

Large-scale chromatofocusing-based method for isolating thymosin β 4 and thymosin β 9 from bovine tissues

A large-scale method for the isolation of thymosin β 4 (up to 120 mg) and thymosin β 9 (up to 40 mg) from bovine lung (up to 2 kg) was developed. The isolation protocol included tissue homogenization in 0.4 M HClO 4, centrifugation, solid-phase extraction through LiChroprep RP-18 material, chromatofocusing on polybuffer exchanger PBE 94-modified Sepharose and dialysis against water. The isolated products were characterized by analytical isoelectric focusing, reversed-phase HPLC, electrospray ionization mass spectrometry and amino acid analysis. The method developed is rapid and convenient, requires no expensive equipment and can be used for the isolation of thymosin β 4 and homologous peptides from various animal tissues.