Exchange Chromatography Research Articles

Multiple-injection technique for isolating a target protein from multicomponent mixtures

An integrated chromatographic process comprising ion exchange (IEC) and hydrophobic interaction chromatography (HIC) for isolating a target protein form multicomponent mixtures has been analyzed. The model mixture contained immunoglobulin G that was the key product of the separation process, cytochrome C and ovalbumin. The adsorption characteristics and the mass transport kinetics of the model proteins have been determined along with their dependencies on the operating variables such as pH, temperature and the salt concentration for IEC as well as HIC media. Limitations of the process efficiency resulting from kinetic effects, solubility constraints and the necessity of the mobile phase exchange between chromatographic steps have been discussed. To improve the performance of the integrated process the multiple-injection technique has been suggested. This technique consisted in loading feed mixtures dissolved in a good solvent onto the column by several small-volume injections under conditions of strong protein adsorption. It allowed diminishing interactions between the sample-solvent and protein and elimination of undesired effects such as band splitting and band broadening. For the process design and optimization a dynamic model has been used accounting for thermodynamics and kinetics of the process. The optimization results indicated superiority of the multiple-injection technique over standard isocratic injections in terms of the process yield and productivity.

LC– and CZE–ICP-MS approaches for the in vivo analysis of the anticancer drug candidate sodium trans-[tetrachloridobis(1H-indazole)ruthenate(iii)] (KP1339) in mouse plasma

Ruthenium-indazole complexes are promising anticancer agents undergoing clinical trials. KP1339 is administered intravenously (i.v.), where serum proteins are the first available biological binding partners. In order to gain a better insight into the mode of action, mice were treated with different doses of KP1339 i.v. and sacrificed at different time points. The blood plasma was isolated from blood samples and analyzed by capillary zone electrophoresis (CZE) and size exclusion/anion exchange chromatography (SEC-IC) both combined on-line to inductively coupled plasma-mass spectrometry (ICP-MS). The performance of the analytical methodology was compared and the interaction of KP1339 with mouse plasma proteins characterized in vivo. Interestingly, the samples of the mice treated with 50 mg kg(-1) and terminated after 24 h showed a ca. 4-fold lowered albumin content and increased ruthenation of albumin aggregates as compared to the untreated control group and the 40 mg kg(-1) group. The majority of Ru was bound to albumin and the stoichiometry of the KP1339 protein binding was determined through the molar Ru/S ratio. In general, good agreement of the data obtained with both techniques was achieved and the SEC-IC method was found to be more sensitive as compared to the CZE-ICP-MS approach, whereas the latter benefits from the shorter analysis time and lower sample consumption.

Identification of new Brazilian allergens from manioc (Manihot esculenta)

Brazil is a very rich country in terms of biodiversity offering broad spectra of possibilities to study new allergens, particularly from foods. In poor and developing countries manioc is a major source of carbohydrates in the diet of millions of people. In Brazil is the ninth cultivated food and the country takes the second place in the world as manioc cultivator. Allergic reactions to manioc were previously reported, but the allergen molecules involved and the cross-reactivity with other plant food sources or latex remain unknown. The aim of present work is to identify new manioc allergens, clone and express them. Based on clinical history, nine patients allergic to manioc were submitted to prick-to-prick test and basophil activation tests and sera were used for immunoblot analysis and ISAC. All patients demonstrated positive IgE reactivity to manioc proteins of approximately 30 and 40 kDa in 1D gel electrophoresis. Using 2D analysis, 6 immunoreactive spots were identified as allergenic-related protein Pt2L4, GADPH and fructose-bisphosphate-aldolase. Three out of five tested sera showed IgE reactivity to Hev b 5 in ISAC. The rich glutamic acid protein, Pt2L4 shows around 40% sequence identity with Hev b5 and 2 of 5 linear IgE epitopes identified for Hev b 5 are partially conserved in Pt2L4. Therefore, the mature sequence was amplified from manioc cDNA using specific primers and cloned into a pET-based expression vector. Clones from two different cultivars were checked and they are all the same sequence but deviate 3 amino acids from the three previously published sequences. Recombinant allergenic-related protein was expressed in E. coli BL21 Star and purified by ion exchange and size exclusion chromatography. Protein will be characterized regarding its physicochemical properties using aminoacid analysis, mass spectrometry, circular dichroism and simulated duodenal digestion. The IgE-binding activity of the recombinant molecule is assessed by IgE immunoblot and ELISA. In addition, IgE cross-reactivity with purified natural Hev b 5 is evaluated using sera of manioc and latex allergic patients.

Isolation, identification and characterization of bacteriocin from Lactobacillus lactis and its antimicrobial and cytotoxic properties

Bacteriocin produced by Lactobacillus lactis strain isolated from the marine environment, showed broad range of antibacterial activity against some major food borne pathogens. Maximum bacteriocin production was observed at 30°C, pH 6.0 and 1.5% sodium chloride solution. The enzymes α-amylase, DNase, RNase and lipase had a positive effect on bacteriocin production. Proteinase K and pepsin strongly inhibited bacteriocin production. Among detergents, Sodium dodecyl sulphate (SDS), Tween 80 and Tritone X-100 stimulated bacteriocin production. EDTA and urea strongly inhibited bacteriocin production. The bacteriocin was purified by ammonium sulphate precipitation and ion exchange (DEAE cellulose) chromatography. The molecular weight was 94 kDA. The purified bacteriocin showed strong cytotoxicity in brine shrimp lethality assay and the LC50 value was 21.54 µg/ml. The study revealed the possibility of using the bacteriocin as a food preservative and the L. lactis strain as a probiotic. Key words: Bacteriocin, Lactobacillus lactis, lactic acid bacteria, cytotoxicity, antagonistic activity.

Identification of the Blue Pigment Formed in aD-Glucose-Glycine Reaction System

D-Glucose (0.7 M), glycine (0.3 M), and sodium hydrogencarbonate (0.1 M) were dissolved in aqueous 30% ethanol at pH 8.0 and left at 50 °C for 4 d in a dark room under nitrogen displacement. The resulting blue pigment was isolated and purified from the blue solution by anionic exchange and gel filtration chromatography. This blue pigment, which is designated Blue-G1, was identified as 5-[1,4-bis-carboxymethyl-5-(2,3,4-trihydroxybutyl)-1,4-dihydropyrrolo[3,2-b]pyrrol-2-ylmethylene]-1,4-bis-carboxymethyl-2-(2,3,4-trihydroxybutyl)-4,5-dihydropyrrolo[3,2-b]pyrrol-1-ium. Blue-G1 had two symmetrical pyrrolopyrrole structures with a trihydroxybutyl group. Blue-G1 had a polymerizing activity, suggesting it to be an important Maillard reaction intermediate through the formation of melanoidins.

Purification and characterization of a new 11S globulin-like protein from Chinese jujube (Ziziphus jujube Mill.) seeds

Around 8 million tons of jujube pomace containing seeds was produced each year in China; so how to utilize these residual matters has been receiving great attentions. The 11S globulins are the major storage proteins, and it can be used as additive agents in many food products with gelling and caking functionality. In the present study, an 11S globulin-like protein (ZSG) was isolated and purified from Chinese jujube (Ziziphus jujube Mill.) seeds by two consecutive anion exchange and size exclusion chromatography. A yield of 8 mg of the ZSG from 1 kg seeds was obtained. The apparent molecular mass of the native ZSG was found to be about 210 kDa. The protein consisted of two subunits with molecular masses of 26.0 kDa and 26.5 kDa, respectively. Amino acid composition analyses indicated that ZSG contained all eight essential amino acids, while lysine was the first limiting amino acids. N-terminal amino acid sequences of 26.0- and 26.5-kDa subunits are GVEETICTLRLLENI and GLEETVCSLRLKENI. The two peptide fragments of 26.0-kDa subunit are ANPDQVLENAFQISR and GVEETICTLR by LC–MS/MS. To the best of our knowledge, this is the first report on the purification of 11S globulin-like protein from Chinese jujube (Ziziphus jujube Mill.) seeds.

Shotgun proteome analysis utilising mixed mode (reversed phase‐anion exchange chromatography) in conjunction with reversed phase liquid chromatography mass spectrometry analysis

The 2-D peptide separations employing mixed mode reversed phase anion exchange (MM (RP-AX)) HPLC in the first dimension in conjunction with RP chromatography in the second dimension were developed and utilised for shotgun proteome analysis. Compared with strong cation exchange (SCX) typically employed for shotgun proteomic analysis, peptide separations using MM (RP-AX) revealed improved separation efficiency and increased peptide distribution across the elution gradient. In addition, improved sample handling, with no significant reduction in the orthogonality of the peptide separations was observed. The shotgun proteomic analysis of a mammalian nuclear cell lysate revealed additional proteome coverage (2818 versus 1125 unique peptides and 602 versus 238 proteins) using the MM (RP-AX) compared with the traditional SCX hyphenated to RP-LC-MS/MS. The MM analysis resulted in approximately 90% of the unique peptides identified present in only one fraction, with a heterogeneous peptide distribution across all fractions. No clustering of the predominant peptide charge states was observed during the gradient elution. The application of MM (RP-AX) for 2-D LC proteomic studies was also extended in the analysis of iTRAQ-labelled HeLa and cyanobacterial proteomes using nano-flow chromatography interfaced to the MS/MS. We demonstrate MM (RP-AX) HPLC as an alternative approach for shotgun proteomic studies that offers significant advantages over traditional SCX peptide separations.

Phytochelatin synthesis plays a similar role in shoots of the cadmium hyperaccumulator Sedum alfredii as in non‐resistant plants

Phytochelatin (PC) synthesis is considered necessary for Cd tolerance in non-resistant plants, but roles for PCs in hyper-accumulating species are currently unknown. In the present study, the relationship between PC synthesis and Cd accumulation was investigated in the Cd hyperaccumulator Sedum alfredii Hance. PCs were most abundant in leaves followed by stems, but hardly detected by the reversed-phase high-performance liquid chromatography (HPLC) in roots. Both PC synthesis and Cd accumulation were time-dependent and a linear correlation between the two was established with about 1:15 PCs : Cd stoichiometry in leaves. PCs were found in the elution fractions, which were responsible for Cd peaks in the anion exchange chromatograph assay. About 5% of the total Cd was detected in these elution fractions as PCs were found. Most Cd was observed in the cell wall and intercellular space of leaf vascular cells. These results suggest that PCs do not detoxify Cd in roots of S. alfredii. However, like in non-resistant plants, PCs might act as the major intracellular Cd detoxification mechanism in shoots of S. alfredii.

Purification of α-Amylase from Bacillus sp. GHA1 and its partial characterization

Bacillus sp. GHA1 was isolated from water samples and screened for the production of α-amylase. Maximum production of amylase by this strain occurs at 42 °C, pH 6.5 and 72 h after cultivation in production medium. The enzyme was purified through successive applications of ammonium sulfate precipitation, ion exchange and hydrophobic interaction chromatography, resulting in a single band with an apparent molecular weight of 66 kDa, as judged by SDS-PAGE. Calcium analysis of the purified enzyme revealed that it contained three metal ions per molecule. The new extracellular α-amylase is active in a wide range of pH with its maximum activity at pH values 5.5–8.0. The optimum temperature for enzyme activity is 57 °C and the presence of calcium has relatively low influence on its activity and thermostability. The Bacillus sp. GHA1 α-amylase with these properties may be suitable for use in detergent and food industries.

Recombination expression and immunoprotective effects of the P6 outer membrane protein of nontypeable Haemophilus influenzae

Objective To clone and express recombinant outer membrane protein P6, determine its optimal expression conditions and to investigate the immunoprotective effects of the P6 protein on mice. Methods The P6 gene of nontypeable Haemophilus influertzae(NTHi) was amplified by PCR from the NTHi genome and cloned into expression vector pET-32a (+) to generate the pET-32a-P6 recombinants. They were confirmed by nuclease digestion and sequence analysis. The verified recombinant was transformed into E. coli BL21 (DE3). Its optimal expression conditions were determined such as engineering strains, the concentration of IPTG, inducing temperature, inducing time, different medium etc. The recombinant protein was purified by Q Sepharose~(TM) XL ion exchange and gel filtration chromatography. The protein was analyzed by SDS-PAGE, Western blot and sequencing. BALB/c mice were immunized with recombinant protein be-fore challenged by NTHi through intraperitoneal injection. Then the mortality rate of different group was com-pared. Results The recombinant P6 of NTHi was successfully constructed and expressed in E. coli at a rel-atively high level. The purity was up to 95% after purification. The relative molecular mass of the protein is 14 145. 848. The recombinant protein was confirmed to show specific reaction on the antiserum through Western blot. The animal experiments showed the mortality rates of immunization groups were significantly lower than that of the control group (P < 0.05). Conclusion The successful expression of the recombinant P6 will be very helpful for the further study on development of vaccine, its purified, immunological activity and antibody preparation. Key words: Nontypeable Haemophilns influenzae; Outer membrane protein; P6; Cloning and expression; Immunoprotective

Molecular cloning, overexpression and characterization of the raw-starch-digesting α-amylase of Bacillus amyloliquefaciens

Raw starch is the most abundant source of glucose in the world. Therefore, finding enzymes capable of digesting raw starch would find high industrial demand. The α-amylase gene of Bacillus amyloliquefaciens ATCC 23842 was amplified, cloned and overexpressed in Escherichia coli BL21 (DE3) cells. The recombinant enzyme was purified to apparent homogeneity using ion exchange and gel filtration chromatography. The raw-starch digestibility of the purified enzyme was characterized by studying the hydrolysis and adsorption rate on a variety of raw starches (potato, cassava, corn, wheat and rice). The raw-starch digestion was further confirmed by scanning electron microscopy studies, which revealed an effective rate of hydrolysis. The kinetic studies revealed a relatively low K m of 2.76 mg/mL, exhibiting high affinity towards the soluble starch as the most preferred substrate and the inhibition kinetic studies revealed a high K i value (350 mM).

Isolation of midgut agglutinin of Culex quinquefasciatus ( Diptera , Culicidae)

A Lectin that agglutinates Eschrechia coli (ATCC 25922) , Staphylococcus aureus (ATCC 6538) live bacteria and various mammalian red blood cells (RBCs) was identified in Culex quinquefasciatus midgut extract ( Cqlec) by using human (three groups: A, B, and O, RH+)mouse, rat, guinea-pig, rabbit and goat erythrocytes. With the use of (NH4)2 SO4 fractionation, anion - exchange and GluNA CBr-Sepharose 6B affinity chromatography, C. quinquefasciatus mid gut lectin has been purified to homogeneity. IEF and reducing SDS/PAGE revealed that the isolated mid gut lectin have isoelectric point (PI) of 6.30, and a subunit approximate molecular weight of 34.5 KDa .The highest agglutination activity of crude and isolated Cqlec were detected against both Eschrechia coli cells and rabbit RBCs. Significant differences in hemagglutinin titers and carbohydrate inhibition were detected between sugar fed and blood fed adult female mosquitoes.Overall agglutinin levels were increased following a blood meal feeding and E. coli induction using a hypodermic needle. This study presents the first report on the occurrence of heterogeneous anti rabbit RBC agglutinins in the midgut extracts of C. quinquefasciatus from Al kharj area in Saudi Arabia. The HA of lectins are Ca 2+ independent, heat-resistant, and are strongly inhibited by D(+)-mannose and D(+) glucose followed by N-acetyl-Dglucosamine. Raffinose and N-acetyl-D-manosamine were found to be moderate inhibitors. Non of the lectins were inhibited by galactose , lactose , trehalose or fetuin (1%) but the glycosubstances mucin and laminarin showed strong inhibitition using low concentrations . 2D-NMR spectroscopy revealed a component of the corresponding residue in structure having group regions of resolution spectrum of Man9- GlcNAc2Asn.

Expression and purification of the recombinant mustard trypsin inhibitor 2 (MTI2) in Escherichia coli

The mustard trypsin inhibitor 2, MTI2, was expressed in Escherichia coli. A specific procedure for its production and purification is described. The recombinant protein was recovered by protein extraction from the insoluble fraction, then renatured and purified by ion exchange and gel filtration chromatography. Finally, the inhibitory activity against trypsin was also determined.

Purification and Classification of Three Lipovitellin Subtypes in the Marbled Sole (Pleuronectes yokohamae)

An immunologlcal analysis using subtype-specific antisera of the major yolk protein lipovltellin (Lv) of the grey mullet (Mugil cephalus) confirmed the presence of the three corresponding Lv subtypes (LvA, LvB, and LvC) in vitellogenic ovaries of the marbled sole (Pleuronectes yokohamae). These three Lv subtypes were purified from sole ovaries by using various combinations of anion exchange, hydroxylapatite, immunoadsorbent, and gel-filtration chromatography. Purified LvA, LvB, and LvC had an apparent native mass of approximately 482, approximately 380, and approximately 372 kDa, respectively, estimated by gel filtration. Analysis of their tertiary structures by SDS-PAGE indicated that LvA, LvB, and LvC were typical of teleost Lvs in having a heavy (H) chain (approximately 105, approximately 102, and approximately 107 kDa, respectively) and a light (L) chain (approximately 22, approximately 19.5, and approximately 25 kDa, respectively). The N-termlnal amino acid (AA) sequences were obtained for the LvA H chain, the LvB H and L chains, and the LvC L chain and compared to the deduced AA sequences of their precursors, vitellogenins (Vgs), in several species. This comparison of LvA, LvB, and LvC with various teleost VgA, VgB, and VgC sequences, respectively, revealed high identities (60-100%). The purified Lv subtypes were subjected to double immunodiffusion using an antiserum against an unclassified Lv of the sole ( Hashimoto et al., 1998 ); only the LvB subtype exhibited immunoreactivity with this antiserum. This result indicates that the previously developed immunoassay using this anti-Lv for the detection of sole Vg is effectively a VgB-specific assay.

Efficient production of active Vibrio proteolyticus aminopeptidase in Escherichia coli by co-expression with engineered vibriolysin

The Vibrio proteolyticus aminopeptidase is synthesized as a preproprotein and then converted into an active enzyme by cleavage of the N-terminal propeptide. In recombinant Escherichia coli, however, the aminopeptidase is not processed correctly and the less-active form that has the N-terminal propeptide accumulates in the culture medium. Recently, we isolated a novel vibriolysin that was expressed as an active form in E. coli by random mutagenesis; this enzyme shows potential as a candidate enzyme for the processing of aminopeptidase. The E. coli cells were engineered to co-express the novel vibriolysin along with aminopeptidase. Co-expression of vibriolysin resulted in an approximately 13-fold increase in aminopeptidase activity, and a further increase was observed in the form lacking its C-terminal propeptide. The active aminopeptidase was purified from the culture supernatant including the recombinant vibriolysin by heat treatment and ion exchange and hydroxyapatite chromatography with high purity and 35% recovery rate. This purified aminopeptidase effectively converted methionyl-human growth hormone (Met-hGH) to hGH. Thus, this co-expression system provides an efficient method for producing active recombinant V. proteolyticus aminopeptidase.

Setaria cervidual specific phosphatase: characterization and its effect on eosinophil degranulation

Setaria cervi, a bovine filarial parasite contains significant acid phosphatase (AcP) activity in its various life stages. Two forms of AcP were separated from somatic extract of adult female parasite using cation exchange, gel filtration and concavalin affinity chromatography. One form having a molecular mass of 79 kDa was characterized as dual specific protein tyrosine phosphatase (ScDSP) based on substrate specificity and inhibition studies. With various substrates tested, it showed significant activity in the order of phospho-L-tyrosine>pNPP>ADP>phospho-L-serine. Inhibition by orthovanadate, fluoride, molybdate, and zinc ions further confirms protein tyrosine phosphatase nature of the enzyme. Km and Vmax determined with various substrates were found to be 16.66 mM, 25.0 microM/ml/min with pNPP; 20.0 mM, 40.0 microM/ml/min with phospho-L-tyrosine and 27.0 mM, 25.0 microM/ml/min with phospho-L-serine. KI with pNPP and sodium orthovanadate (IC50 33.0 microM) was calculated to be 50.0 mM. Inhibition with pHMB, silver nitrate, DEPC and EDAC suggested the presence of cysteine, histidine and carboxylate residues at its active site. Cross-reactivity with W. bancrofti-infected sera was demonstrated by Western blotting. ScDSP showed elevated levels of IgE in chronic filarial sera using ELISA. Under in vitro conditions, ScDSP resulted in increased effector function of human eosinophils when stimulated by IgG, which showed a further decrease with increasing enzyme concentration. Results presented here suggest that S. cervi DSP should be further studied to determine its role in pathogenesis and the persistence of filarial parasite.

Soybean β-conglycinin constituent subunits: Isolation, solubility and amino acid composition

Abstract A protocol to purify soybean β-conglycinin constituent subunits (α, α′, β) from soybean ( Glycine max Linn. Merr.) was investigated. The combination of anion exchange chromatograph and Immobilized Metal Ion Affinity Chromatography (IMAC), under 6 M urea was used. The subunits were evaluated by SDS–PAGE, Differential Scanning Calorimetry (DSC) and Size Exclusion Chromatography (SEC). The subunits α, α′ and β exhibited isoelectric precipitation at pH 4.0–5.0, 4.0–5.0 and 4.0–6.0, respectively. β subunit exhibited constant solubility around 56% at pH ⩾ 6. Total Essential Amino Acid (TEAA) and Essential Amino Acid Index (EAAI) revealed that both TEAA and EAAI of α′ subunits were lower than α and β subunits. This new fractionation method has practical significance for the researchers to prepare enough suitable amounts of samples to study the structure–physicochemical function relationships of β-conglycinin constituent subunits, and to supply enough diet for animal experiments to study the physiological function of single β-conglycinin constituent subunit.

Isolation, Purification, And Characterization Of Some Cystein Proteases From Bovine Mastites Milk

Proteolytic activity of cysteine proteases were studied in bovine mastitis milk, four fractions designated as F1,F2,F3,F4 with cysteine protease activity were separated from leukocytes cell by ion- exchange Chromatography through DEAE-Cellulose The most active fraction F4 was selected for further purification utilizing gel filtration Chromatography on Sephadex G-100 column it has been found that F4 most likely being cathepsin B. purification folds and the enzyme yield was 46.66 and 31.81% respectively . polyacrylamide gel electrophoresis test indicated that the enzyme has been purified to homogeneity by giving a single band . The results of enzyme characterization showed that the molecular weights were 31000 and 30000 Daltons as determined by gel filtration and electrophoresis methods in present of reducing agent SDS- PAGE respectively. The optimum pH for the enzyme activity was 6.0 and it was stable at pH values ranged between 4.5 - 6.5. The enzyme exhibited the maximum activity at 45ºC and the enzyme retained its entire activity over 30 min incubation at 30 -50 C and it retained (50, 20, 10) % of its entire activites over 30 min incubation at (60, 70, 80) C respectively. From this results and results observed from the effect of inhibitor and activator reagents we suggest that enzyme F4 possibly belonged to cathepsin B.

Structural Studies with Polysaccharides from Sterculia urens and Sterculia lychnophora

Monosaccharide analysis of the deacetylated polysaccharide from Sterculia urens was performed by thin-layer chromatography and by High-pH-Anion Exchange (Dionex) chromatography.[...]

Purification of Caudal-Related Homeodomain Transcription Factor and Its Binding Characterization

Human CDX2 is known as a caudal-related homeodomain transcription factor that is expressed in the intestinal epithelium and is important in differentiation and maintenance of the intestinal epithelial cells. The caudal-related homeobox proteins bind DNA according to a helix-turn-helix structure, thereby increasing the structural stability of DNA. A cancer-tumor suppressor role for Cdx2 has been shown by a decrease in the level of the expression of Cdx2 in colorectal cancer but the mechanism of transcriptional regulation has not been examined at the molecular level. We developed a large-scale system for expression of the recombinant, novel CDX2, in the Escherichia coli. A highly purified and soluble CDX2 protein was obtained in E. coli strain BL21(DE3)RIL and a hexahistidine fusion system using Ni-NTA affinity column, anion exchange, and gel filtration chromatography. The identity and secondary structure of the purified CDX2 protein were confirmed by MALDI-TOF MS, Western blot, and a circular dichroism analyses. In addition, we studied the DNA binding activity of recombinant CDX2 by ELISA experiment and isolated human CDX2 binding proteins derived from rat cells by an immobilized GST-fusion method. Three CDX2-binding proteins were found in the gastric tissue, and those proteins were identified to the homeobox protein Hox-D8, LIM homeobox protein 6, and SMC1L1 protein.