Isolation, purification and characteristics of R-phycoerythrin from a marine macroalga Heterosiphonia japonica

R-phycoerythrin is one of the three phycobiliproteins which are extensively employed as fluorescent probes, and it is prepared from red macroalgae. Phycobiliproteins in the marine red macroalga Heterosiphonia japonica were extracted in 50 mM phosphate buffer (pH 7.0) and precipitated by salting-out. The R-phycoerythrin was isolated by gel filtration with Sepharose CL-4B and Sephadex G-200. Then it was purified by ion exchange chromatography on DEAE Sepharose Fast Flow which was developed by linear ionic strength gradients. The purified R-phycoerythrin gave a ratio of A₅₆₅ to A₂₈₀ of 4.89. It showed a single band and a pI of 4.8 on the examination by polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing. The polypeptide analysis of the purified R-phycoerythrin by SDS–PAGE demonstrated that it contains four chromophore-carrying subunits and no colorless polypeptide and has two hexameric aggregates. The preparative procedures of the R-phycoerythrin purification established based on the experiments exhibit advantages and can offer a reference for R-phycoerythrin preparation from other marine red macroalga.     

Cloning, expression, purification, and characterization of a designer protein with repetitive sequences

An artificial protein containing alternating hydrophilic–hydrophobic blocks of amino acids was designed in order to mimic the structure of synthetic multiblock copolymers. The hydrophobic block consisted of the six amino acids Ala Ile Leu Leu Ile Ile (AILLII) and the hydrophilic block of the eight amino acids Thr Ser Glu Asp Asp Asn Asn Gln (TSEDDNNQ). The coding DNA sequence of the cluster was inserted into an commercial pET 30a(+) vector using a two step strategy. The expression of the artificial protein in Escherichia coli was optimized using a temperature shift strategy. Only at cultivation temperature of 24 °C after induction expression was observed, whereas at 30 and 37 °C no target protein could be detected. Cells obtained from a 15 L bioreactor cultivation of E. coli were disintegrated by mechanical methods. Interestingly, glass bead milling and high pressure homogenization resulted in a different solubility of the target protein. The further purification was carried out by affinity chromatography using the soluble homogenized protein. Extreme conditions (6 M urea, 0.5 M NaCl) were applied in order to prevent aggregation to insoluble particles. The designer protein showed an extremely high tendency to form dimers or trimers caused by intermolecular interactions which were even not broken under the conditions of SDS–polyacrylamide gel electrophoresis, rendering the behavior during purification different from proteins usually found in nature. The protein preparation was not completely pure according to SDS–PAGE stained by Coomassie blue or silver. In MALDI-TOF-MS, nano-ESI qTOF-MS of the entire protein preparation and nano-ESI-MS after digestion by trypsin and chymotrypsin impurities were not detectable.     

Identification and partial purification of DnaK homologue from extremely halophilic archaebacteria, Halobacterium cutirubrum

The levels of synthesis of six proteins were increased at elevated growth temperature of the extremely halophilic archaebacterium Halobacterium cutirubrum. One of these proteins, with an apparent molecular mass of 97 kDa on sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE), bound to an ATP-agarose column in the presence of 4 M NaCl, but not in the absence of salt, indicating that this protein retained its ATP-binding activity only at high salt concentration. The NH₂-terminal sequence of this protein and the internal sequences of the tryptic peptides covering 1/3 of the total number of residues coincided with that deduced from the nucleotide sequence of the dnaK gene isolated from H. cutirubrum. The results strongly suggest that this apparent 97-kDa protein is the gene product of dnaK, although the molecular mass calculated from the nucleotide sequence is only 68,495, much smaller than the value of this protein determined by SDS–PAGE. Ferguson plot analysis indicated that this protein showed anomalous mobility on SDS–PAGE. We have purified DnaK homologue to greater than 90% homogeneity with stepwise elution from an ATP-agarose column.     

Simple high-performance liquid chromatographic purification procedure for porcine plasma haptoglobin

Haptoglobin is an acute-phase protein and its plasma levels increase consistently in response to infection and inflammation. Some evidence has demonstrated that haptoglobin is involved in the immune responses. In this study, we established a novel high-performance liquid chromatographic purification procedure for porcine plasma haptoglobin. The procedure required an ammonium sulfate fractionation and a HPLC Superose 12 gel-permeation chromatography. Purified porcine haptoglobin possessed one heavy (β) and light chain (α) on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, under reducing conditions, with a M_r (molecular mass) of about 42 000 and 14 000 for heavy (β) and light chains (α), respectively. Although the N-terminal amino acid sequence of porcine heavy chain of haptoglobin has never been reported previously, the analyses of N-terminal amino acid sequence showed a great sequence similarity to that of human and other animal species. In addition, Western blot using our specific antibody prepared against porcine M_r 42 000 chain did react with human haptoglobin and likewise, the antibody against human haptoglobin also cross-reacted with purified porcine M_r 42 000 chain. Thus, it confirmed that the identity of the porcine protein purified from our procedures was as haptoglobin.     

Characterization of Staphylococcus species by SDS–PAGE of Whole-Cell and Extracellular Proteins

In this study, a total of fifteen staphylococcal strains belonging to different species were characterized by whole-cell and extracellular protein profiles using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The results are presented as dendrograms after quantitative analysis of the band patterns with a computer program. Visual inspection of protein bands and cluster analysis of protein patterns of to be used 15 strains, representing 10 Staphylococcus species, showed that whole-cell and extracellular protein profiles differed in several protein bands in Staphylococcus aureus, S. epidermidis, S. simulans, and other species of Staphylococcus; however, the differences were insufficient for reliable differentiation of Staphylococcus species by the SDS–PAGE method.     

Sodium dodecyl sulfate-agarose gel electrophoresis for the detection and isolation of amyloid curli fibers

Curli are amyloid-like fibers on the surface of some strains of Escherichia coli and Salmonella enteritidis. We tested the use of horizontal sodium dodecyl sulfate (SDS)–agarose gel electrophoresis to detect, isolate, and quantitate curli. Cell extracts fractionated in SDS–agarose gels and stained with Coomassie blue exhibited a soluble fraction that entered the gel and an insoluble fraction that remained in the well. Much more insoluble material was observed with curli-proficient strains than with strains that do not make curli. Both highly purified curli and the insoluble material isolated from an SDS–agarose gel could be dissociated into monomers when treated with formic acid. For quantitation, we immobilized samples in SDS–agarose prior to electrophoresis. This avoids losses during the staining of the gel. Our methods provide a rapid and simple fractionation of curli using equipment that is readily available.     

Sequencing of Gel-Isolated Proteins Using Microblotter Capillary Liquid Chromatography-Electrospray Mass Spectrometry

Enzymatic digests of proteins isolated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) were separated by capillary high-performance liquid chromatography (HPLC). The column eluate was split to an electrospray mass spectrometer on one side and to both a UV detector and a microblotter on the other side. Using the microblotter, the peptides eluted from the column were collected directly onto a polyvinylidene difluoride (PVDF) membrane for Edman sequencing. Thus, a peptide mass map from the mass spectrometric analysis and a prepared PVDF membrane for subsequent Edman sequencing were generated in a single experiment. The addition of molecular mass information to the blotted LC eluate is useful for determining the most important peaks to undergo Edman sequencing. Coupling the capillary HPLC with a microblotter to electrospray mass spectrometry provides an integrated system for separation, collection, and structural analysis of protein digests. It provides high levels of sensitivity, recovery, and convenience for protein characterization. Proteins loaded onto SDS–PAGE at low picomole levels can be analyzed by the new integrated system.     

Various forms of mouse lactoferrins: purification and characterization

This work was conducted to study the microheterogeneity of mouse lactoferrin (LF). Two forms, LF₁ and LF₂, could be purified from uterine luminal fluid by ion-exchange HPLC on a Protein PAK SP 5PW column. Another form, LF₃, was purified from the epididymis homogenate by affinity chromatography on a column of Protein A-Sepharose coupled with the purified LF₂ antibody that was prepared to give no crossreaction with serum albumin. Both LF₁ and LF₂ showed a M_r 74 000 band while LF₃ gave a M_r 70 000 band on reducing SDS–PAGE. All of them were reduced to a M_r 68 000 band after they had been digested with N-glycosidase F. The data from automated Edman degradation confirmed the completely identical 19 amino acid sequences in the N-terminal regions of these three LFs, except the lack of N-terminal Lys–Ala of LF₂/LF₃ in LF₁. LF in tissue homogenates was immunodetected by Western blot procedure using the purified LF₂ antibody. Different amounts of LF with a molecular mass of the 70 000 or 74 000 were distributed in the non-sexual organs such as kidney, spleen, lung, heart and liver and the sexual glands including epididymis, vagina, uterus, ovary and prostate. No LF was detected in stomach, intestine, testis and seminal vesicle.     

A method for the purification of Shiga-like toxin 1 subunit B using a commercially available galabiose–agarose resin

We describe a procedure for the affinity purification of Shiga toxin 1 subunit B (SLTB) using a commercial galabiose–agarose resin. Recombinant SLTB was purified to 99% homogeneity in a single-step protocol, from the periplasmic extracts of Vibrio cholerae 0395 N1/pSBC54. SDS–PAGE of the affinity purified SLTB showed one band of 8 kDa MW. SLTB purified by this procedure retained its chemical and biological activity as demonstrated by re-binding to the galabiose–agarose resin, and receptor-mediated binding and uptake in Vero cells. The galabiose–agarose resin could isolate roughly 1 mg of SLTB/mL of gel. The resin was stable over 3 years and 500 cycles/year of usage. Hence, this method is a straightforward approach to the large-scale preparation of SLTB at a reasonable cost.     

Keratinolytic activity of purified alkaline keratinase produced by Scopulariopsis brevicaulis (Sacc.) and its amino acids profile

Sodium dodecyl sulfate–polyacrlyamide gel electrophoresis (SDS–PAGE) was used to assess the purity and molecular weight of the previously purified alkaline keratinase enzyme of Scopulariopsis brevicaulis. The enzyme was homogenous, as seen by a single band of protein, and had an apparent molecular weight of 28.5 kDa. Amino acid profile of the purified keratinase revealed that it was composed of 14 different amino acids with high proportions of glutamic acid (20.86%), alanine (14.52%), glycine (14.21%), leucine (8.59%) and serine (7.81%). The enzyme contained moderate amounts of valine (6.01%), threonine (5.58%) and phenyl alanine (5.22%). The purified enzyme of S. brevicaulis exerted a potent keratinolytic activity and was capable to hydrolyze different keratinaceous materials with highest activity on chicken feathers followed by human nails and human hair.     

Purification of a growth inhibitor for Ehrlich ascites mammary carcinoma cells from bovine mammary gland

A growth inhibitor for Ehrlich ascites mammary carcinoma cells in vitro has been purified from bovine mammary gland. The purification procedure involving homogenization and differential centrifugation under hypotonic conditions, ammonium sulfate precipitation, ultrafiltration, gel chromatography and preparative polyacrylamide gel electrophoresis (PAGE) yielded an inhibitor showing half-maximal inhibition of cell proliferation in concentrations of 1–3 ng protein per ml. Upon ¹²⁵I labelling and analysis by SDS gel electrophoresis, most purified preparations revealed a single band of 12–14 kD, likely to be representative for the inhibitory protein. The inhibitor was shown to affect resumption of proliferation of stationary cells; however, it was inactive towards cells stimulated by incubation with medium before adding the inhibitor. The inhibitor is heat-labile, does not act by exhausting essential components of culture medium, and its action is antagonized by insulin.     

A highly thermostable,alkaline CMCase produced by a newly isolated Bacillus sp. VG1

An extracellular, highly thermostable and alkaline CMCase was purified from Bacillus sp. VG1 using ion exchange and gel filtration chromatography. Enzyme was optimally produced in a medium containing 1.0% CMC and 0.5% tryptone. The purified CMCase had a pH optimum of 9–10 and a half life of 12 min even at 100 °C. The enzyme activity was reduced by Hg²⁺ and stimulated by Co²⁺, Na⁺ and K⁺. Various detergents and proteinases moderately inhibited the CMCase activity. The molecular weight studies showed a single band on SDS–PAGE.     

Lytic enzyme complex of an antagonistic Bacillus sp. X-b: isolation and purification of components

Bacillus sp. X-b, a biocontrol agent against certain plant pathogenic fungi, secretes a complex of hydrolytic enzymes, composed of chitinase, chitosanase, laminarinase, lipase and protease. Homogenized mycelium of basidiomycete Macrolepiota procera induced activities of these enzymes more effectively than colloidal chitin or partially purified cell walls of another basidiomycete Polyporus squamosus. Subjected to a multi-step purification, the specific activity of chitinase increased 36-fold, chitosanase 69-fold, lipase 44-fold and laminarinase 15-fold. Partially purified chitinase showed two major bands with molecular masses of 46 000 and 35 000 on sodium dodecyl sulfate–polyacrylamide gel electrophoresis while chitosanase and lipase appeared as single bands with molecular masses of 27 000 and 62 000, respectively.     

Expression and Purification of the BmK M1 Neurotoxin from the Scorpion Buthus martensii Karsch

The gene encoding a neurotoxin (BmK M1) from the scorpion Buthus martensii Karsch was expressed in Saccharomyces cerevisiae at a high level with the alcohol dehydrogenase promoter. SDS–PAGE of the culture confirmed expression and showed secretion into medium from yeast. Recombinant BmK M1 was purified rapidly and efficiently by ion exchange and gel filtration chromatography to homogeneity, produced a single band on tricine–SDS–PAGE, and processed the homologous N-terminus. Amino acid analysis and N-terminal sequencing demonstrated that the recombinant toxin was processed correctly from the α-mating factor leader sequence and was chemically identical to the native form. The expressed recombinant BmK M1 was toxic for mice, which indicated that it was biologically active. Quantitative estimation showed that recombinant BmK M1 had an LD₅₀ similar to that of the native toxin.     

Improving sodium dodecyl sulfate polyacrylamide gel electrophoresis detection of low-abundance protein samples by rapid freeze centrifugation

This work presents a rapid and simple freeze centrifugation method to concentrate dilute protein solutions for detection by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) Coomassie blue staining. Moreover, a simple way to assemble a cryoconcentration device is presented, and its use is discussed. Commercial purified protein standard and an enzyme with high fructosyltransferase (FTase) activity, coming from target fractions obtained by chromatographic separation, were used as an example. FTase, coming directly from the chromatographic fractions, was difficult to view through SDS–PAGE analysis; however, it was easily visualized, and its activity was enhanced, after the application of the freeze centrifugation protocol presented here.     

Purification of α-amylase from Bacillus licheniformis by chromatofocusing and gel filtration chromatography

A combination of chromatofocusing and gel filtration chromatography resulted in a simple purification of -amylase from Bacillus licheniformis. The purification was approximately 77-fold. Identification of the purity was established by SDS–PAGE. Molecular weight and isoelectric point of the purified enzyme were 58 kDa and 7.18 respectively. Western blot analysis confirms the specificity of antibody raised against purified -amylase.     

Purification and characterization of bovine mannan-binding protein

Bovine mannan-binding protein (bMBP) was observed in serum byits Ca²⁺ -dependent binding to mannan and by an M_rof 28 kDaunder reducing conditions on sodium dodecyl sulphate—polyacrylamidegel electrophoresis (SDS—PAGE). The lectin was isolatedby precipitation with polyethyl-eneglycol (PEG), affinity chromatographyon mannan—Sepharose eluted with EDTA, and absorption onSepharose 4B rabbit anti-bovine Ig to remove anti-mannan antibodies.Fractions containing the lectin were reapplied to mannan—Sepharoseand eluted first with N-acetyl-D-glucosamine (GlcNAc) to removeconglutinin, and then with mannose to elute the 28 kDa lectin.Further purification was achieved by ion-exchange chromatographyon Mono-Q and by man-nose-gradient elution from a mannan-Sepharosecolumn. SDS—PAGE of the purified lectin showed three highmolecular weight bands under non-reducing conditions. The reducedprotein gave a single band of 28 kDa. On gel permeation chromatographyunder non-dissociating conditions, the protein emerged at avolume corresponding to M_r     

Rapid purification and properties of human glycodelin (endometrial α2-globulin)

The method presented can easily produce milligram amounts of glycodelin from pregnancy endometrium, with a 19% yield. It involves anion-exchange chromatography, gel permeation and chromatofocusing; it results in one stainable band at M_r 28 000 after sodium dodecyl sulphate–polyacrylamide electrophoresis, as well as after immunoblot analysis, performed using an affinity-purified IgG fraction from an antiserum against glycodelin. In spite of this, the corresponding gel isoelectric focusing pattern gives four stainable bands with pI values between 4.55 and 5.2. Western immunoblot analysis of tissue extracts indicates the presence of glycodelin epitopes associated with materials heavier than the native protein. Circular dichroism spectra of the highly purified protein in water solutions indicate a large amount of β-sheet conformation, whereas those obtained with different proportions of 2-propanol in water, show an increased proportion of α-helix conformation.     

Capillary gel electrophoresis: separation of major erythrocyte membrane proteins

A new separation method of human erythrocyte membrane proteins by sodium dodecyl sulfate capillary gel electrophoresis (SDS–CGE) is described. In this method, a replaceable gel matrix was used. Seven major erythrocyte membrane proteins, α-and β-spectrin, ankyrin 2.1, band 3 (anion-exchanger), 4.1a and b, and 4.2 (pallidin), were separated and identified by SDS–CGE method. High reproducible migration times of these proteins (inter-assay coefficients of variation less than 2%), as well as quantification (inter-assay coefficients of variation less than 11%) were obtained. This new SDS–CGE method may provide important diagnostic evidence for hereditary spherocytosis. It can be a powerful diagnostic tool in place of SDS polyacrylamide gel electrophoresis for erythrocyte membrane protein analysis.     

Purification of Porcine Phospholamban Expressed inEscherichia coli

Phospholamban (PLB) is a small hydrophobic protein that regulates contractility in the heart. This membrane protein expressed in bacterial cells is resistant to purification by conventional strategies that have been used to isolate expressed soluble proteins. Therefore, in order to obtain both wild-type and mutant PLB proteins, we have amplified the PLB gene by the polymerase chain reaction from genomic DNA of porcine heart and inserted it into the pGEX-2T plasmid expression vector. In this vector, the gene product fused to glutathioneS-transferase has been expressed in JM109Escherichia colicells. The expressed fusion protein was found associated predominantly with insoluble cellular constituents. However, most of the fusion protein was readily extracted with SDS. PLB was subsequently purified by a simple procedure consisting of isolation of the fusion protein by preparative SDS–gel electrophoresis, followed by a second electrophoretic separation of PLB after its cleavage from the fusion protein by thrombin. This isolation method yields 3–4 mg of PLB per liter of cells, in a form which is capable of functional interaction with the Ca-ATPase in reconstituted proteoliposomes.