Two-step Ion-exchange Chromatography Research Articles

Expression, purification, characterization and crystallization of Panax quinquefolius ginsenoside glycosyltransferase Pq3-O-UGT2

Pq3-O-UGT2, derived from Panax quinquefolius, functions as a ginsenoside glucosyltransferase, utilizing UDP-glucose (UDPG) as the sugar donor to catalyze the glycosylation of Rh2 and F2. An essential step in comprehending its catalytic mechanism involves structural analysis. In preparation for structural analysis, we expressed Pq3-O-UGT2 in the Escherichia coli (E. coli) strain Rosetta (DE3). The recombinant Pq3-O-UGT2 was purified through Ni-NTA affinity purification, a two-step ion exchange chromatography, and subsequently size-exclusion chromatography (SEC). Notably, the purified Pq3-O-UGT2 showed substantial activity toward Rh2 and F2, catalyzing the formation of Rg3 and Rd, respectively. This activity was discernible within a pH range of 4.0–9.0 and temperature range of 30–55 °C, with optimal conditions observed at pH 7.0–8.0 and 37 °C. The catalytic efficiency of Pq3-O-UGT2 toward Rh2 and F2 was 31.43 s−1 mΜ−1 and 169.31 s−1 mΜ−1, respectively. We further crystalized Pq3-O-UGT2 in both its apo form and co-crystalized forms with UDPG, Rh2 and F2, respectively. High-quality crystals were obtained and X-ray diffraction data was collected for all co-crystalized samples. Analysis of the diffraction data revealed that the crystal of Pq3-O-UGT2 co-crystalized with UDP-Glc belonged to space group P1, while the other two crystals belonged to space group P212121. Together, this study has laid a robust foundation for subsequent structural analysis of Pq3-O-UGT2.

Production of functional human nerve growth factor from the submandibular glands of mice using a CRISPR/Cas9 genome editing system.

Nerve growth factor (NGF) is an essential trophic factor for the growth and survival of neurons in the central and peripheral nervous systems. For many years, mouse NGF (mNGF) has been used to treat various neuronal and non-neuronal disorders. However, the biological activity of human NGF (hNGF) is significantly higher than that of mNGF in human cells. Using the CRISPR/Cas9 system, we constructed the transgenic mice expressing hNGF specifically in their submandibular glands. As demonstrated by fluorescence immunohistochemical staining, these mice produced hNGF successfully, with 0.8mg produced per gram of submandibular glands. hNGF with 99% purity was successfully extracted by two-step ion-exchange chromatography and one-step size-exclusion chromatography from the submandibular glands of these transgenic mice. Further, the purified hNGF was verified by LC-MS/MS. We analyzed the NH2-terminus of hNGF using both Edman degradation and LC-MS/MS-based methods. Both results showed that the obtained hNGF lost the NH2-terminal octapeptide (SSSHPIFH). Moreover, the produced hNGF demonstrated a strong promotion in the proliferation of TF1 cells.

Comparison of purified and crude extracted coagulants from plant species for turbidity removal

The coagulation properties of crude extracts and purified proteins of seeds of Moringa stenopetala and tubers of Maerua subcordata, which are locally used for purification of turbid water in Ethiopia, were studied. The turbidity removal effectiveness was compared using synthetic turbid and natural river water where the initial turbidity of synthetic water was approximately 260 NTU, whereas, for river water, the initial turbidity was 30 NTU. The active coagulants from the extracts were purified by the single-step and the two-step ion exchange (IEX) chromatography technique using 0.1, 0.3 and 0.6 M concentrations of NaCl solutions. The protein quantification of a single-step purified active coagulant eluted using 0.6 M showed that the protein content of the M. stenopetala extract was 1016 µg/mL, whereas the concentration of the M. subcordata extract was 700 µg/mL. The dose for coagulation using crude extracts and purified proteins of M. subcordata on surface water was 4 and 2 mg/L, respectively, with 85% of coagulation activity. In the case of M. stenopetala, 14 mg/L dose of crude extracts, which was seven times higher than that of the purified protein, was needed to obtain the desired residual turbidity (5 NTU). The results also indicated that in the two-step purified coagulants, the sludge volume was reduced compared to crude extracts and alum. In conclusion, active coagulants from seeds of M. stenopetala could be scaled up and used in water treatment technology, whereas purifying the extracts from M. subcordata is not required as the crude extracts performed equally well.

Proton-induced production and radiochemical isolation of 44Ti from scandium metal targets for 44Ti/44Sc generator development

Scandium-44g (half-life 3.97h) shows promise for application in positron emission tomography (PET), due to favorable decay parameters. One of the sources of 44gSc is the 44Ti/44gSc generator, which can conveniently provide this radioisotope on a daily basis at a diagnostic facility. Titanium-44 (half-life 60.0 a), in turn, can be obtained via proton irradiation of scandium metal targets. A substantial 44Ti product batch, however, requires high beam currents, long irradiation times and an elaborate chemical procedure for 44Ti isolation and purification. This study describes the production of a combined 175MBq (4.7mCi) batch yield of 44Ti in week long proton irradiations at the Los Alamos Isotope Production Facility (LANL-IPF) and the Brookhaven Linac Isotope Producer (BNL-BLIP). A two-step ion exchange chromatography based chemical separation method is introduced: first, a coarse separation of 44Ti via anion exchange sorption in concentrated HCl results in a 44Tc/Sc separation factor of 102–103. A second, cation exchange based step in HCl media is then applied for 44Ti fine purification from residual Sc mass. In summary, this method yields a 90–97% 44Ti recovery with an overall Ti/Sc separation factor of ≥106.

Purification and assembling a fused capsid protein as an enterovirus 71 vaccine candidate from inclusion bodies to pentamer-based nanoparticles

An efficient preparation process for a novel Enterovirus 71 (EV71) vaccine was developed in this paper, which is a fused antigen by connecting the truncated capsid proteins of VP1, VP2 and VP3 into one molecule through flexible peptide linkers and expressed in E. coli as inclusion bodies. The fused protein was purified at denatured state through two-step ion exchange chromatography, with final purity above 95%, host cell proteins below 0.003% and residual DNA less than 50ng/mL. During the following refolding and assembling process through dilution, the fused antigen precipitated completely, while the precipitation was efficiently inhibited with 2M urea or 0.5M arginine as an additive. Size exclusion chromatography analysis indicated the protein formed soluble aggregates with linear or rod-like appearance in transmission electron microscopy. These aggregates transformed into pentamers with a size of 15nm at pH 8.0 after the additive removing. Moreover, most of the pentamers assembled as sphere-like particulates about 25–40nm after being induced by calcium chloride. High antigen-specific IgG titer was elicited by immunization with the nanoparticles in mouse model. Splenocytes proliferative responses and cytokines analysis indicated this particulate antigen could induce humoral and cellular immune responses. These results lay foundations for developing the fused antigen as an alternative vaccine against hand-foot-and-mouth disease (HFMD) and for the large-scale production for E.coli-based vaccines.

Expression of Two Novel β-Glucosidases from Chaetomium atrobrunneum in Trichoderma reesei and Characterization of the Heterologous Protein Products.

Two novel GH3 family thermostable β-glucosidases from the filamentous fungus Chaetomium atrobrunneum (CEL3a and CEL3b) were expressed in Trichoderma reesei, purified by two-step ion exchange chromatography, and characterized. Both enzymes were active over a wide range of pH as compared to Neurospora crassa β-glucosidase GH3-3, which was also expressed in T. reesei and purified. The optimum temperature of both C. atrobrunneum enzymes was around 60°C at pH 5, and both enzymes had better thermal and pH stability and higher resistance to metallic compounds and to glucose inhibition than GH3-3. They also showed higher activity against oligosaccharides composed of glucose units and linked with β-1,4-glycosidic bonds and moreover, had higher affinity for cellotriose over cellobiose. In hydrolysis tests against Avicel cellulose and steam-exploded sugarcane bagasse, performed at 45°C, particularly the CEL3a enzyme performed similarly to N. crassa GH3-3 β-glucosidase. Taking into account the thermal stability of the C. atrobrunneum β-glucosidases, they both represent promising alternatives as enzyme mixture components for improved cellulose saccharification at elevated temperatures.

Production of Influenza Virus HA1 Harboring Native-Like Epitopes by Pichia pastoris.

The outbreak of the H5N1 highly pathogenic avian influenza which exhibits high variation had brought a serious threat to the safety of humanity. To overcome this high variation, hemagglutinin-based recombinant subunit vaccine with rational design has been considered as a substitute for traditional virion-based vaccine development. Here, we expressed HA1 part of the hemagglutinin protein using the Pichia pastoris expression system and attained a high yield of about 120mg/L through the use of fed-batch scalable fermentation. HA1 protein in the culture supernatant was purified using two-step ion-exchange chromatography. The resultant HA1 protein was homogeneous in solution in a glycosylated form, as confirmed by endoglycosidase H treatment. Sedimentation velocity tests, silver staining of protein gels, and immunoblotting were used for verification. The native HA1 reacted well with conformational, cross-genotype, neutralizing monoclonal antibodies, whereas a loss of binding activity was noted with the denatured HA1 form. Moreover, the murine anti-HA1 serum exhibited a virus-capture capability in the hemagglutination inhibition assay, which suggests that HA1 harbors native-like epitopes. In conclusion, soluble HA1 was efficiently expressed and purified in this study. The functional glycosylated protein will be an alternative for the development of recombinant protein-based influenza vaccine.

Separation and Analysis of Boron Isotope in High Plant by Thermal Ionization Mass Spectrometry.

Knowledge of boron and its isotope in plants is useful to better understand the transposition and translocation of boron within plant, the geochemical behavior in the interface between soil and plant, and the biogeochemical cycle of boron. It is critical to develop a useful method to separate boron from the plant for the geochemical application of boron and its isotope. A method was developed for the extraction of boron in plant sample, whose isotope was determined by thermal ionization mass spectrometry. The results indicated that this method of dry ashing coupled with two-step ion-exchange chromatography is powerful for the separation of boron in plant sample with large amounts of organic matters completely. The ratios of boron isotope composition in those plant tissue samples ranged from −19.45‰ to +28.13‰ (total range: 47.58‰) with a mean value of 2.61 ± 11.76‰ SD. The stem and root isotopic compositions were lower than those in flower and leaf. The molecular mechanism of boron isotope may be responsible for the observed variation of boron isotopic composition and are considered as a useful tool for the better understanding of boron cycling process in the environment and for the signature of living systems.

Facile purification of Escherichia coli expressed tag-free recombinant human tumor necrosis factor alpha from supernatant

Fusing affinity tag at N-terminus was reported to decrease the biological activity of the recombinant human tumor necrosis factor alpha. Although preparation of tag-free rhTNF-α has already been achieved, the processes were yet laborious, especially in large scale. In this paper, tag-free rhTNF-α was almost equally synthesized by Escherichia coli in both soluble and insoluble forms. A two-step ion exchange chromatography, DEAE-Sepharose combined with CM-Sepharose, was developed to purify the soluble specie from supernatant after cell lysis. Native PAGE and HP-SEC showed the rhTNF-α extracted from supernatant existed in a homogeneous form. HP-SAX and SDS–PAGE analysis demonstrated the purity of the final fraction was over 98% with a very high recovery of 75%. Circular dichroism spectrum demonstrated that β-sheet structure was dominant and fluorescence analysis suggested no dramatic exposure of aromatic amino acid residues on the protein surface. Bioassay indicated that purified rhTNF-α was biologically active with a specific activity of approximately 2.0×107U/mg. All these results suggested that this two-step ion exchange chromatography is efficient for preparation of biologically active tag-free rhTNF-α from supernatant.

Isolation of three novel Antarctic psychrotolerant feather-degrading bacteria and partial purification of keratinolytic enzyme from Lysobacter sp. A03

Three psychrotolerant bacteria (designated as A03, A08 and A17U) were isolated from penguin feathers collected in the Elephant Island, Antarctic. They were able to grow in feather meal as sole carbon and nitrogen source at 9 °C, 20 °C and 30 °C. Based on 16S rDNA sequencing, the strains were identified as belonging to the genera Lysobacter, Arthrobacter and Chryseobacterium, respectively. The isolate Lysobacter sp. A03 was capable to degrade feather meal almost completely in 7 days of cultivation at 20 °C, which was also the optimal growth temperature for Arthrobacter sp. A08 and Chryseobacterium sp. A17U. Despite the optimum growth temperature of the isolates was in the range of psychrotolerant microorganisms, the enzyme extracts showed maximum caseinolytic and keratinolytic activities under temperatures between 15 and 20 °C above that point, which is expected when enzymes are isolated from their source organism. For Lysobacter sp. A03, proteolytic activity was strongly inhibited by serine protease inhibitor PMSF, while for Arthrobacter sp. A08 and Chryseobacterium sp. A17U the enzymatic activity was inhibited by EDTA and partially inhibited by PMSF and 1,10-phenantroline. These results were in agreement with those found in zymograms. The protease of Lysobacter sp. A03, partially purified by two-step ion exchange chromatography, was only inhibited by PMSF. The peptide bands were analyzed by mass spectrometry resulting in the absence of significant homology to other Lysobacter proteins or microbial proteases of NCBI database. The production of keratinolytic proteases by these cold-adapted bacteria can be valuable for the reduction of energy costs in the application of enzymes in industrial processes as an alternative to mesophilic and thermophilic microorganisms.

Inhibition of Tumor Growth in a Mouse Xenograft Model by the Humanized Anti-HGF Monoclonal Antibody YYB-101 Produced in a Large-Scale CHO Cell Culture

The humanized anti-hepatocyte growth factor (HGF) monoclonal antibody (mAb) YYB-101 is a promising therapeutic candidate for treating various cancers. In this study, we developed a bioprocess for large-scale production of YYB-101 and evaluated its therapeutic potential for tumor treatment using a xenograft mouse model. By screening diverse chemically defined basal media formulations and by assessing the effects of various feed supplements and feeding schedules on cell growth and antibody production, we established an optimal medium and feeding method to produce 757 mg/l of YYB-101 in flask cultures, representing a 7.5-fold increase in titer compared with that obtained under non-optimized conditions. The optimal dissolved oxygen concentration for antibody production was 70% pO2. A pH shift from 7.2 to 7.0, rather than controlled pH of either 7.0 or 7.2, resulted in productivity improvement in 5 L and 200 L bioreactors, yielding 737 and 830 mg/ml of YYB-101, respectively. The YYB-101 mAb highly purified by affinity chromatography using a Protein A column and two-step ion exchange chromatography effectively neutralized HGF in a cell-based assay and showed potent tumor suppression activity in a mouse xenograft model established with human glioblastoma cells.

Reduced Antigenicity of Recombinant Ovine Factor VIII in Hemophilia A Inhibitor Patient Plasmas

AbstractAbstract 1124Hemophilia A is an X-linked recessive disorder caused by deficiencies or functional defects in coagulation factor VIII (fVIII). Approximately 20–30% of patients with severe hemophilia A develop antibodies against fVIII (inhibitors) following fVIII replacement therapy, which presents significant complication to the control of subsequent bleeding episodes. State of the art treatment options for patients with inhibitors include fVIII-bypassing agents such as recombinant factor VIIa or activated prothrombin-complex concentrate. Previously, plasma-derived porcine fVIII was a treatment option for inhibitor patients and was effective due to the reduced antigenicity of porcine fVIII toward anti-human fVIII inhibitors. However due to concerns regarding viral contamination, the plasma-derived porcine fVIII products were discontinued and no alternative fVIII products have been made available to patients with inhibitors. Presently, a recombinant porcine fVIII product (OBI-1, Inspiration Biopharmaceuticals) is being investigated in two phase 3 clinical trials for congenital and acquired hemophilia A. Rationale for the development of such a product consists of the prior success of plasma-derived porcine fVIII and the concept that the most effective and lowest risk treatment for fVIII deficiency, even in the presence of inhibitors, remains a fVIII product. Recently, a line of hemophilia A sheep was reestablished from banked frozen sperm and the ovine fVIII (ofVIII) gene, causal mutation, and protein were genetically and biochemically characterized. B-domain deleted (BDD) ovine fVIII shares 86% identity to human fVIII at the amino acid level and confers phenotypic correction, in vivo, to hemophilia A mice using a tail transaction bleeding model. Recombinant ofVIII was expressed in baby-hamster kidney cells and purified to > 95% homogeneity using a two-step ion exchange chromatography procedure. Highly purified ofVIII displays a specific activity of 18,300 units/mg, which is approximately twice that of recombinant BDD human fVIII. Furthermore, the decay of ofVIII activity following thrombin activation is slower than BDD human fVIII suggesting prolonged activity in vivo. Lastly, ofVIII demonstrates equivalent binding to human von Willebrand factor at physiological concentrations in vitro. A translational aim of the present study was to test the hypothesis that unique sequences within ofVIII confer differential antigenicity compared to human and/or porcine fVIII in congenital and acquired inhibitor patient plasmas. To address this hypothesis, the reactivity of 28 samples (22 congenital patient samples designated 1–22, and 6 acquired hemophilia A patient samples designated A1-A6) from the Emory IRB approved inhibitor bank towards recombinant BDD human, porcine, and ovine fVIII were assessed by enzyme-linked immunosorbant assay (Figure 1). When normalized to the reactivity towards human fVIII, the data revealed reduced reactivity towards ofVIII in 27 of 28 total samples. In only one patient was the reactivity towards ofVIII greater than that towards human fVIII and, in this sample, the reactivity towards porcine fVIII also was greater than 100%. Furthermore, plasma reactivity to ovine fVIII was significantly reduced compared to porcine fVIII (P = 0.025; Mann-Whitney U Test). Median values of the relative cross reactivity towards porcine and ovine fVIII were 54 and 38%, respectively. Preliminary inhibitor analysis (Bethesda assay) of three samples shown to contain titers against human fVIII of 25, 19, and 68 BU/ml, revealed undetectable inhibitor titers towards ofVIII in 2 samples, and a titer of 5 BU/ml in the third, respectively. These results suggest that additional orthologous recombinant fVIII molecules may be enabling to the treatment of patients harboring pathogenic inhibitors to human fVIII. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

OPTIMAL PRODUCTION OF L-threo-2,3-DIHYDROXYPHENYLSERINE (L-threo-DOPS) ON A LARGE SCALE BY DIASTEREOSELECTIVITY-ENHANCED VARIANT OF L-THREONINE ALDOLASE EXPRESSED IN Escherichia coli

This study examined the efficient production and optimal separation procedures for pure L-threo-3,4-dihydroxyphenylserine (L-threo-DOPS) from a mixture of diastereomers synthesized by whole-cell aldol condensation reaction, harboring diastereoselectivity-enhanced L-threonine aldolase in Escherichia coli JM109. The addition of the reducing agent sodium sulfite was found to stimulate the production of L-threo-DOPS without affecting the diastereoselectivity ratio, especially at the 50 mM concentration. The optimal pH for diastereoselective synthesis was 6.5. The addition of Triton X-100 also strongly affected the synthesis yield, showing the highest conversion yield at a 0.75% concentration; however, the diastereoselectivity of the L-threonine aldolase was not affected. Lowering the temperature to 10°C did not significantly affect the diastereoselectiviy without affecting the synthesis rate. At the optimized conditions, a mixture of L-threo-DOPS and L-erythro-DOPS was synthesized by diastereoselectivity-enhanced L-threonine aldolase from E. coli in a continuous process for 100 hr, yielding an average of 4.0 mg/mL of L-threo-DOPS and 60% diastereoselectivity (de), and was subjected to two steps of ion exchange chromatography. The optimum separation conditions for the resin and solvent were evaluated in which it was found that a two-step process with the ion-exchange resin Dowex 50 W × 8 and activated carbon by washing with 0.5 N acetic acid was sufficient to separate the L-threo-DOPS. By using two-step ion-exchange chromatography, synthesized high-purity L-threo-DOPS of up to 100% was purified with a yield of 71%. The remaining substrates, glycine and 3,4-dihydroxybenzaldehyde, were recovered successfully with a yield of 71.2%. Our results indicate this potential procedure as an economical purification process for the synthesis and purification of important L-threo-DOPS at the pharmaceutical level.

Development and Characterization of Recombinant Ovine Factor VIII

Abstract 1193Factor VIII (fVIII) is an essential glycoprotein procofactor in the blood coagulation cascade. Mutations in the fVIII gene often result in diminished plasma fVIII activity causing the bleeding disorder hemophilia A, which obeys X-linked recessive genetics and affects approximately 1 in 7500 males. Current treatment is limited to intravenous infusion of plasma-derived or recombinant human (h)-fVIII containing products. This therapy is expensive and requires multi-weekly injections to achieve prophylaxis, which must be maintained for the duration of the patients’ life to avoid debilitating joint disease as well as life-threatening bleeding episodes. While gene therapy is being explored as a potential cure, much research is aimed at improving the therapeutic utility of recombinant fVIII. Our laboratory has been interested in the comparison of recombinant fVIII molecules derived from different animal species and has identified many species-specific biochemical properties. The characterization of recombinant murine (m)-factor VIII revealed near complete stability at physiologic concentrations following thrombin activation upon which h-fVIII activity decays on the order of minutes. In contrast, porcine (p)-fVIII demonstrates 100-fold increased post-translational biosynthesis over h-fVIII as well as decreased engagement of the endoplasmic reticulum-resident unfolded protein response. Arruda and colleagues showed that canine (c)-fVIII displays 3-fold higher specific activity than that of h-, p-, or m-fVIII and currently is being used to treat bleeding episodes in canine hemophilia A colonies. Finally, we have generated hybrid fVIII constructs, mapped the sequences necessary and sufficient for certain differential properties, identified immunogenic epitopes inherent to each species and created novel fVIII molecules with combined potentially beneficial characteristics. The current study is a continuation of this line of research focusing on ovine (o)-fVIII. Recently, a line of hemophilia A sheep was re-established and the pathology and clinical profile was described. O-fVIII possesses a high degree of homology to h-fVIII with a similar A1-A2-activation peptide-A3-C1-C2 domain structure. The causative mutation was identified as a single nucleotide insertion causing a frameshift and premature stop codon in exon 14. Administration of human and hybrid h/p-fVIII in these sheep corrected the bleeding phenotype transiently, but invariably induced the formation of high titer anti-fVIII inhibitory antibodies leading to premature mortality. Herein, we describe the generation, expression and biochemical characterization of recombinant full-length and B-domain-deleted (BDD) o-fVIII. O-fVIII was cloned into the mammalian expression vector, ReNeo and a pace/furin linker sequence was introduced between the A2 domain and the activation peptide. Utilizing a baby hamster kidney cell expression system, full-length o- and h-fVIII demonstrated similar expression levels at 0.098 ± 0.024 and 0.022 ± 0.006 units/106 cells/24hr, respectively (p = 0.99). Removal of the ovine B domain resulted in increased expression (0.97 ± 0.2 units/106 cells/24hr, p < 0.001) to a level equivalent to BDD h-fVIII (0.49 ± 0.149 units/106 cells/24hr; p = 0.06). BDD o-fVIII was purified to virtual homogeneity from conditioned serum-free media using a two-step ion-exchange chromatography procedure identified previously for the purification of recombinant h- and p-fVIII. Two independent preparations were analyzed and determined to have specific activities of 12,300 and 14,760 units/mg. SDS-PAGE analysis revealed three predominant polypeptides species, with a minority of intact single chain as well as predominantly processed heavy and light chains. Uniquely, the heavy and light chain polypeptides displayed similar relative mobility and could only be distinguished by thrombin proteolysis or immuno-precipitation prior to SDS-PAGE using monoclonal antibodies with known epitopes in either the heavy or light chains. We anticipate that recombinant o-fVIII, similar to the situation that has occurred with the canine hemophilia A colony and recombinant c-fVIII, will facilitate the maintenance of the ovine hemophilia A herd and their utilization as a relevant large animal model for research and development of novel hemophilia A therapeutics. Disclosures:No relevant conflicts of interest to declare.

Gene Cloning and Heterologous Expression of a Novel Endoglucanase, Swollenin, fromTrichoderma pseudokoningiiS38

The coding sequence of a novel cellulolytic factor, swollenin, was isolated from the cellulolytic fungus Trichoderma pseudokoningii S38. The full-length swo2 gene encodes a protein of 494 amino acids with a calculated molecular mass of 51,393 Da, which includes a putative 22-amino-acid signal peptide. Sequence analysis revealed significant identity between isolated swollenin and that from Trichoderma reesei. The swollenin gene was further expressed and purified in T. reesei QM9414. The expressed swollenin protein was consequently purified by two-step ion exchange chromatography. The purified swollenin had subtle hydrolytic activities on xylan and yeast cell wall glucan, while no apparent activities on carboxymethy cellulose, cotton fiber, filter paper, or cellulose powder CF11 were observed. These results indicate that although swollenin maintains unidentified glycohydrolytic activities, it is inactive against beta-1,4-glycosidic bonds in cellulose. Its exact role in lignocellulose hydrolysis calls for further analysis.

Leukocyte 12‐lipoxygenase: expression, purification and physical characterization

The expression and purification procedure [Richards, K.M. and Marnett, L.J. (1997) Biochemistry 36, 6692–6699] for leukocyte 12‐lipoxygenase was updated for the production of the large quantities of protein necessary for the characterization of its physical properties. A two‐step FPLC ion exchange chromatography procedure was adequate for purification as evaluated by SDS PAGE, specific activity measurement, isoelectric focusing, and electrospray ionization mass spectrometry. The solution properties of the enzyme were examined by variable temperature circular dichroism spectroscopy, differential scanning microcalorimetry, and dynamic light scattering measurements. The redox characteristics of the active site iron atom were determined by EPR spectroscopy following treatment with a variety of fatty acid hydroperoxides. Leukocyte 12‐lipoxygenase was found to be more similar to reticulocyte 15‐lipoxygenase than to soybean lipoxygenase‐1 or human 5‐lipoxygenase.(Supported by the National Institutes of Health GM 62140)

Human Calprotectin: Effect of Calcium and Zinc on its Secondary and Tertiary Structures, and Role of pH in its Thermal Stability

Calprotectin, a heterodimeric complex belonging to the S100 protein family, has been found predominantly in the cytosolic fraction of neutrophils. In the present study, human calprotectin was purified from neutrophils using two-step ion exchange chromatography. The purified protein was used for circular dichroism study and fluorescence analysis in the presence of calcium and zinc at physiological concentrations, as well as for assessment of its inhibitory activity on the K562 leukemia cell line. The thermal stability of the protein at pH 7.0 (physiological pH) and 8.0 (similar to intestinal pH) was also compared. The results of cell proliferation analysis revealed that human calprotectin initiated growth inhibition of the tumor cells in a dose-dependent manner. The intrinsic fluorescence emission spectra of human calprotectin (50 microg/ml) in the presence of calcium and zinc ions show a reduction in fluorescence intensity, reflecting a conformational change within the protein with exposure of aromatic residues to the protein surface that is important for the biological function of calprotectin. The far ultraviolet-circular dichroism spectra of human calprotectin in the presence of calcium and zinc ions at physiological concentrations show a decrease in the alpha-helical content of the protein and an increase in beta- and other structures. Our results also show that increasing the pH level from 7.0 to 8.0 leads to a marked elevation in the thermal stability of human calprotectin, indicating a significant role for pH in the stability of calprotectin in the gut.

Simple and Rapid Methods for Purification and Characterization of Active Coagulants from the Seeds of Vigna unguiculata and Parkinsonia aculeata

The coagulating properties of aqueous crude extracts and purified proteins of Vigna unguiculata and Parkinsonia aculeata seeds, which are traditional water coagulants in rural areas of Tanzania, were studied. The coagulation activity assays were done using one millilitre (ml) of kaolin water samples. Coagulating proteins were purified in two-step ion exchange chromatography. The properties of coagulant protein were compared with Moringa oleifera. Coagulating components eluted by 0.6 M NaCl in both coagulants are cationic proteins that have the molecular mass of about 6 kDa, which is very similar to that of M. oleifera. The proteins of V. unguiculata and P. aculeata eluted by 0.3 M NaCl also harbour coagulation activity but proteins eluted with 0.6 M NaCl have higher activity. The dosage for coagulation using purified proteins of both coagulants is about 5 to 10 times lower than that of crude seed extracts. The optimum floc settling time of water treated by crude seed extracts and purified proteins ranged between two and two and half hours. Coagulating proteins of both coagulants eluted by 0.6 M NaCl are thermoresistant and retained coagulation activity of 87% to 92% after boiling for two hours at 80 °C and one hour at 95°C. Thermotolerant proteins of V. unguiculata eluted by 0.6 M NaCl and P. aculeata have wider pH range of 5.5 to 8.5 for coagulation activity than those of M. oleifera proteins. The present investigation reveals the possibility of using purified natural coagulants for water treatment to produce safe drinking water.

Two-step ion-exchange chromatographic purification of recombinant hirudin-II and its C-terminal-truncated derivatives expressed in Pichia pastoris

The scene of the protein micro-heterogeneity of recombinant hirudin-II (HV2) expressed in Pichia pastoris was investigated. It was shown that three derivatives of HV2 were present in the fermentation broth of P. pastoris, which were intact HV2 and its two derivatives truncated the C-terminal amino acid residue Gln and Leu-Gln, respectively. To purify the minor degradation derivatives of HV2, a simple, biocompatible and scale-up-feasible purification process with two-step ion-exchange chromatography was established instead of usual reverse phase chromatography. The purities of end products were over 96% and the residual endotoxin less than 0.5 EU/ml.

Purification, Characterization, Kinetic Properties, and Thermal Behavior of Extracellular Polygalacturonase Produced by Filamentous Fungus Tetracoccosporium sp.

For the first time, a polygalacturonase from the culture broth of Tetracoccosporium sp. was isolated and incubated at 30 degrees C in an orbital shaker at 160 rpm for 48 h. The enzyme was purified by ammonium sulfate precipitation and two-step ion-exchange chromatography and had an apparent molecular mass of 36 kDa, as shown by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Its optimum activity was at pH 4.3 and 40 degrees C, and the Km and Vmax values of this enzyme (for polygalacturonic acid) were 3.23 mg/mL and 0.15 micromol/min, respectively. Ag+, Co2+, EDTA, Tween-20, Tween-80, and Triton X-100 stimulated polygalacturonase activity whereas Al3+, Ba2+, Ca2+, Fe2+, Fe3+, Ni2+, Mg2+, Mn2+, and SDS inhibited it. In addition, iodoacetamide and iodoacetic acid did not inhibit enzyme activity at a concentration of 1 mM, indicating that cysteine residues are not part of the catalytic site of polygalacturonase. We studied the kinetic properties and thermal inactivation of polygalacturonase. This enzyme exhibited a t1/2 of 63 min at 60 degrees C and its specific activity, turnover number, and catalytic efficiency were 6.17 U/mg, 113.64 min-1, and 35.18 mL/(min.mg), respectively. The activation energy (DeltaE#) for heat inactivation was 5.341 kJ/mol, and the thermodynamic activation parameters DeltaG#, DeltaH#, and DeltaS# were also calculated, revealing a potential application for the industry.