Size Exclusion Chromatography Purification Research Articles

Immunomodulatory Activity In Vitro and In Vivo of a Sulfated Polysaccharide with Novel Structure from the Green Alga Ulvaconglobata Kjellman

Algae accumulate large amounts of polysaccharides in their cell walls or intercellular regions. Polysaccharides from algae possess high potential as promising candidates for marine drug development. In this study, a sulfated polysaccharide, UCP, from the green alga Ulva conglobata Kjellman was obtained by water extraction, anion-exchange, and size-exclusion chromatography purification, and its structure was characterized by a combination of chemical and spectroscopic methods. UCP mainly consisted of →4)-α/β-l-Rhap-(1→, →4)-β-d-Xylp-(1→ and →4)-β-d-GlcAp-(1→ residues. Sulfate ester groups were substituted mainly at C-3 of →4)-l-Rhap-(1→ and C-2 of →4)-β-d-Xylp-(1→. Partial glycosylation was at C-2 of →4)-α-l-Rhap-(1→ residues. UCP possessed a potent immunomodulatory effect in vitro, evaluated by the assays of lymphocyte proliferation and macrophage phagocytosis. The immunomodulatory activity of UCP in vivo was further investigated using immunosuppressive mice induced by cyclophosphamide. The results showed that UCP markedly increased the spleen and thymus indexes and ameliorated the cyclophosphamide-induced damage to the spleen and thymus. UCP could increase the levels of white blood cells, lymphocytes, and platelets, and improve the hematopoietic inhibition caused by cyclophosphamide. Moreover, UCP significantly promoted the secretions of the immunoglobulin (Ig)G, IgE, and IgM. The data demonstrated that UCP is a novel sulfated polysaccharide and may be a promising immunomodulatory agent.

Analysis of the regenerative capacity of human serum exosomes after a simple multistep separation from lipoproteins.

Due to the abundance of lipoproteins in blood, it is challenging to characterize the biological functions and components of blood-derived extracellular vesicles. The aim of this study was to develop a multiple-step purification protocol to separate serum exosomes from serum proteins and lipoproteins and assess their regenerative potential. Exosomes were isolated by concentrating them in human serum using ultracentrifugation (UC), followed sequentially by density gradient (DG) UC and size exclusion chromatography (SEC). Purity and characterization were assessed by western blots, Lipoprint®, enzyme-linked immunosorbent assay, electron microscopy, mass spectrometry, and nanoparticle tracking analysis. Functionality was assessed by cell proliferation analysis and with an in vivo subcutaneous angiogenesis model. SEC alone isolated nano-sized vesicles possessing vesicle markers TSG101 and CD9, but there was a substantial presence of apolipoprotein B, predominantly derived from very-low- and intermediate-density lipoprotein particles. This was reduced to an undetectable level using the combined UC DG SEC approach. Mass spectrometry identified 224 proteins in UC DG SEC isolates relative to the 135 from SEC, with considerable increases in exosome-related proteins and reductions in lipoproteins. A consistent but limited increase in human dermal fibroblast proliferation and evidence of neovascularization enhancement were observed after exposure to UC DG SEC exosomes. An UC DG SEC purification protocol considerably improved the removal of lipoproteins during isolation of serum exosomes. The purified exosomes stimulated cell proliferation and potentially increased an in vivo angiogenic response. This multistep purification allows for more accurate identification of serum exosome functional activity and composition.

Development of triblock polymersomes for catalase delivery based on quality by design environment

Catalase is an antioxidant enzyme with potential application in photoprotection, skin aging, and skin disorders as vitiligo. However, as a large and hydrophilic molecule, catalase topical application is hampered by poor skin penetration. In this sense, nanostructured drug delivery systems such as polymersomes (polymeric vesicles) can significantly promote the enzyme access to skin. Since the concept of designing quality in early stages of biotechnological products development is a regulatory demand over the world, in this study, we employed Quality by Design (QbD) principles to design catalase-loaded polymersomes for topical products. QbD and Design of Experiments (DoE) approaches were used to investigate the impact of stirring speed (SS =750 and 1500 rpm), stirring time (ST = 24 and 48 h), and concentration of catalase (CAT = 0.48 and 0.24 mg/mL) in pharmaceutical development critical quality attributes of PEO5PPO68PEO5 polymersomes for topical application. The design space counterplot of hydrodynamic diameter (200 ≤ HD ≤ 400), encapsulation efficiency (2≤ %EE ≤ 5), polydispersity index (0.1 ≤ PDI ≤ 0.3), as a function of ST and CAT indicated that polymersomes with optimized characteristics could be developed with 0.24 ≥ CAT ≤ 0.40; 40 ≥ ST ≤ 48 and SS= 800 rpm. Encapsulation efficiency (%EE) was determined directly by size exclusion chromatography purification of the polymersomes, and the highest value was %EE = 4.72 ± 0.07 %, nonetheless catalase as an enzyme is effective at very low concentrations. The results point out time and cost-saving strategies that could serve as an initial step to develop a robust industrial process for the production of catalase-loaded topical polymersomes.

Development of BioRad NGC and GE ÄKTA pure systems for highly automated three column protein purification employing tandem affinity, buffer exchange and size exclusion chromatography

Affinity purification, such as Protein A (ProA) followed by size exclusion chromatography (SEC) remains a popular method to obtain research scale proteins. With the need for higher throughput protein production increasing for discovery research, there is substantial interest in the automation of complex protein purification processes, which often start with a ProA step followed by SEC. However, the harsh elution conditions from ProA based chromatography can destabilize some proteins resulting in particulates, which in turn can cause column fouling and potential cross-contamination of subsequent purifications. We modified both Bio Rad NGC and ÄKTA Pure systems to run a three-column process (ProA to buffer exchange to SEC) enabling automated tandem affinity to SEC purification while minimizing the risk of SEC column fouling and subsequent cross-contamination. The intervening buffer exchange column, unlike the final SEC column, can be rapidly regenerated using harsh methods between runs, and these automated systems are capable of processing up to six samples per day without user intervention.

CGMP production of astatine-211-labeled anti-CD45 antibodies for use in allogeneic hematopoietic cell transplantation for treatment of advanced hematopoietic malignancies.

The objective of this study was to translate reaction conditions and quality control methods used for production of an astatine-211(211At)-labeled anti-CD45 monoclonal antibody (MAb) conjugate, 211At-BC8-B10, from the laboratory setting to cGMP production. Five separate materials were produced in the preparation of 211At-BC8-B10: (1) p-isothiocyanato-phenethyl-closo-decaborate(2-) (B10-NCS), (2) anti-CD45 MAb, BC8, (3) BC8-B10 MAb conjugate, (4) [211At]NaAt, and (5) 211At-BC8-B10. The 211At-labeling reagent, B10-NCS, was synthesized as previously reported. BC8 was produced, then conjugated with B10-NCS under cGMP conditions to form BC8-B10. [211At]NaAt was produced by α-irradiation of Bi targets, followed by isolation of the 211At using a “wet chemistry” method. The clinical product, 211At-BC8-B10, was prepared by reacting [211At]NaAt with BC8-B10 in NH4OAc buffer (pH 5.5) for 2 min at room temperature, followed by size-exclusion chromatography purification. Quality control tests conducted on the 211At-BC8-B10 included evaluations for purity and identity, as well as pyrogen and sterility tests. Stability of the 211At-BC8-B10 in 25 mg/mL sodium ascorbate solution was evaluated at 1, 2, 4, 6 and 21 h post isolation. For qualification, three consecutive 211At-BC8-B10 clinical preparations were successfully conducted in the cGMP suite, and an additional cGMP clinical preparation was carried out to validate each step required to deliver 211At-BC8-B10 to a patient. These cGMP preparations provided 0.80–1.28 Gbq (21.5–34.5 mCi) of 211At-BC8-B10 with radiochemical purity of >97%. The preparations were found to be sterile and have a pyrogen level <0.50 EU/mL. Cell binding was retained by the 211At-BC8-B10. 211At-BC8-B10 in ascorbic acid solution demonstrated a radiochemical stability of >95% for up to 21 h at room temperature. The experiments conducted have defined conditions for translation of 211At-BC8-B10 production from the laboratory to cGMP suite. This study has allowed the initiation of a phase I/II clinical trial using 211At-BC8-B10 (NCT03128034).

Ultrasonic extraction, structural characterization, physicochemical properties and antioxidant activities of polysaccharides from bamboo shoots (Chimonobambusa quadrangularis) processing by-products

In this study, a novel polysaccharide (PCPs) was obtained from bamboo shoots (Chimonobambusa quadrangularis) processing by-products through the ultrasonic-assisted extraction (UAE) and Sephadex G-100 size-exclusion chromatography purification. By using a Box–Behnken design (BBD) to optimize the parameters for extracting the crude PCPs with UAE, the maximum yield of crude PCPs (8.76%) was obtained under the optimal extraction conditions as follows: ratio of water to raw material 20.2mL/g, extraction temperature 49°C, ultrasonic power 240W, and extraction time 40min. Structurally, PCPs was identified as hetero-polysaccharides, which was composed of Man, Rha, GlcA, GalA, Glc, Gal, Xyl, and Ara in a molecular ratio of 1.00:2.05:2.28:2.36:15.76:49.06:16.70:39.86. The molecular weight distribution analysis showed that PCPs had three components with molecular weights of 136.1, 29.7 and 8.3kD, respectively. The glycosidic linkage analysis showed that PCPs contained 1→or 1→6 glycosidic linkages (35.1%), 1→2 or 1→4 glycosidic linkages (14.8%), and 1→3 glycosidic linkages (50.1%). conformations. The obtained results revealed that PCPs with a semi-crystalline structure exhibited important functional properties including the repose angle, swelling index (SC), water retention capacity (WRC), and oil retention capacity (ORC). The viscoelastic property of PCPs system exhibited a gel-like behavior with storage modulus G′ always being higher than loss modulus G″. PCPs displayed excellent thermal stability as assessed by rheology and TGA/DSC. Furthermore, PCPs exhibited good chemical antioxidant activities in DPPH, ABTS and hydroxyl radical scavenging abilities, and metal chelating activity.

Characterization of a novel polysaccharide isolated from Rosa roxburghii Tratt fruit and assessment of its antioxidant in vitro and in vivo

In this study, a novel water-soluble polysaccharide (RRTP1-1) was obtained from Rosa roxburghii Tratt fruit through the ultrasonic-assisted extraction followed by anion-exchange and size-exclusion chromatography purification. The primary structure of RRTP1-1 was investigated by UV, FT-IR, HPLC, GC−MS, 1H and 13C NMR spectroscopy. Chemical composition analysis revealed that RRTP1-1 had an average molecular weight of 97.58kDa and consisted of Man, Rha, GlcA, GalA, Glc, Gal, Ara and Xyl in a molar ratio of 2.88:1.39:2.83:1.00:69.11:3.04:2.52:3.41. This polysaccharide was proven to be a kind of glucan, owning a backbone structure of →6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→. Congo red test indicated that RRTP1-1 had no triple helix structure. Antioxidation tests performed in vitro revealed that RRTP1-1 possessed obviously DPPH, hydroxyl and superoxide radicals scavenging activities. Furthermore, antioxidant assays in vivo showed that RRTP1-1 at 200 or 400mg/kg dose could significantly enhance the activities of antioxidant enzymes (CAT, SOD, and GSH-Px), increase TAOC values, and decrease the LPO and MDA levels in different degrees in the serum of D-Gal aging-induced mice. The present results suggested that RRTP1-1 could be a new source of natural antioxidants for applications in functional foods and dietary supplemental products.

Dissociation and purification of the endogenous membrane-bound Vo complex from Pichia pastoris

Most proteins occur and function in complexes rather than as isolated entities in membranes. In most cases macromolecules with multiple subunits are purified from endogenous sources. In this study, an endogenous membrane-protein complex was obtained from Pichia pastoris, which can be grown at high densities to significantly improve the membrane protein yield. We successfully isolated the membrane-bound Vo complex of V-ATPase from P. pastoris using a fusion FLAG tag attached to the C-terminus of subunit a to generate the vph-tag strain, which was used for dissociation and purification. After FLAG affinity and size exclusion chromatography purification, the production quantity and purity of the membrane-bound Vo complex was 20 μg l−1 and >98%, respectively. The subunits of the endogenous membrane-bound Vo complex observed in P. pastoris were similar to those obtained from S. cerevisiae, as demonstrated by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Therefore, successful dissociation and purification of the membrane-bound Vo complex at a high purity and sufficient quantity was achieved via a rapid and simple procedure that can be used to obtain the endogenous membrane-protein complexes from P. pastoris.

Conversion of turbot skin wastes into valuable functional substances with an eco-friendly fermentation technology

Booming development of fishing economy has significantly accelerated the fish waste generation in the world. The disposal of fish waste poses a serious environmental problem. The fish waste contains substantial amount high quality protein, which is an excellent resource of bioactive substance. In the conscious of eco-friendly and economical disposal, converting these waste into high value-added products for industrial applications is of profound implication. In this study, microorganism fermentation was employed and developed for the high-value utilization of discarded turbot skin. Among the various strains, Aspergillus oryzae showed the most promising hydrolysis ability. Under optimal conditions, a comprehensive detailed description of the fermentation process of hydrolysate from Aspergillus oryzae (AOH) is described. Additionally, amino acid compositions, volatile compounds, and antioxidant activity of AOH are studied. Size exclusion chromatography purification could effectively enhance the antioxidant activity. During gastrointestinal digestion, excellent residual antioxidant efficacy can be achieved by AOH and its chelating activity was improved. In physicochemical (temperature, light, pH) stability assays, AOH demonstrated better antioxidative stability in a wide range of pH and temperature conditions, but exhibited sensitivity to sunlight exposure. The results pointed out that turbot skin can be a cheap resource of antioxidant, which has many promising applications in pharmaceutical and food industry for health promotion and food preservation. Finally, fermentative bioprocess could be identified as a clean production which can not only be used for minimizing the impact of environment but also converting fish wastes into valuable functional substances.

Considerations for Sample Preparation Using Size-Exclusion Chromatography for Home and Synchrotron Sources.

The success of a SAXS experiment for structural investigations depends on two precise measurements, the sample and the buffer background. Buffer matching between the sample and background can be achieved using dialysis methods but in biological SAXS of monodisperse systems, sample preparation is routinely being performed with size exclusion chromatography (SEC). SEC is the most reliable method for SAXS sample preparation as the method not only purifies the sample for SAXS but also almost guarantees ideal buffer matching. Here, I will highlight the use of SEC for SAXS sample preparation and demonstrate using example proteins that SEC purification does not always provide for ideal samples. Scrutiny of the SEC elution peak using quasi-elastic and multi-angle light scattering techniques can reveal hidden features (heterogeneity) of the sample that should be considered during SAXS data analysis. In some cases, sample heterogeneity can be controlled using a small molecule additive and I outline a simple additive screening method for sample preparation.

Two-step purification strategy for enhanced recovery of recombinant hepatitis B surface antigen from Pichia pastoris

ABSTRACTUnivariate screening on factors affecting the purification performance of recombinant hepatitis B surface antigen (HBsAg) on ion exchange chromatography (IEC) and size exclusion chromatography (SEC) and the establishment of a two-step purification strategy were performed. Amongst four IEC adsorbents examined, the use of Q Sepharose XL IEC adsorbent under optimized conditions together with optimized SEC purification was able to efficiently purify HBsAg. An established purification strategy comprising the two techniques further demonstrated adaptability for scale-up operations with a final total purification factor (PF) of 94.82 ± 16.20, HBsAg purity of 95.48% and recovery yield of 78.07%.

Extraction and Isolation of Antineoplastic Pristimerin from Mortonia greggii (Celastraceae)

The aim of this research was to identify, extract and isolate pristimerin in leaves, stems and roots of the Mexican plant Mortonia greggii (Celastraceae). The principal objective was to determine the best laboratory experimental conditions for the extraction and isolation of this powerful natural anticancer agent from the root tissue. Six experimental factors in solid-liquid pristimerin extraction were analyzed: solvent systems, number of extractions, ratio of plant weight (g)/solvent volume (mL) used, time of extraction, temperature and agitation. A mathematical model was generated for pristimerin purity and yield. Ethanol, first extraction, 0.5 ratio of plant weight/solvent volume (g/mL), 0.5 h, 200 rpm and 49.7°C were optimal conditions for the extraction of this phytochemical. The degree of purification of pristimerin root extract was studied by size-exclusion chromatography (SEC) using Sephadex LH-20 reaching fractions with purification indexes (PI) greater than 2 and recoveries of 28.3%. When fractions with purification indices higher than 1 and less than 2 were accumulated, the recovery of pristimerin increased by about 73.6%. By combining the optimum extracts and SEC purification protocols, an enriched fraction containing 245.6 mg pristimerin was obtained from 100 g of root bark, representing about 14.4%, w/w, pristimerin from the total solids presented in the fraction.

Semi-Automated Hydrophobic Interaction Chromatography Column Scouting Used in the Two-Step Purification of Recombinant Green Fluorescent Protein

BackgroundHydrophobic interaction chromatography (HIC) most commonly requires experimental determination (i.e., scouting) in order to select an optimal chromatographic medium for purifying a given target protein. Neither a two-step purification of untagged green fluorescent protein (GFP) from crude bacterial lysate using sequential HIC and size exclusion chromatography (SEC), nor HIC column scouting elution profiles of GFP, have been previously reported.Methods and ResultsBacterial lysate expressing recombinant GFP was sequentially adsorbed to commercially available HIC columns containing butyl, octyl, and phenyl-based HIC ligands coupled to matrices of varying bead size. The lysate was fractionated using a linear ammonium phosphate salt gradient at constant pH. Collected HIC eluate fractions containing retained GFP were then pooled and further purified using high-resolution preparative SEC. Significant differences in presumptive GFP elution profiles were observed using in-line absorption spectrophotometry (A395) and post-run fluorimetry. SDS-PAGE and western blot demonstrated that fluorometric detection was the more accurate indicator of GFP elution in both HIC and SEC purification steps. Comparison of composite HIC column scouting data indicated that a phenyl ligand coupled to a 34 µm matrix produced the highest degree of target protein capture and separation.ConclusionsConducting two-step protein purification using the preferred HIC medium followed by SEC resulted in a final, concentrated product with >98% protein purity. In-line absorbance spectrophotometry was not as precise of an indicator of GFP elution as post-run fluorimetry. These findings demonstrate the importance of utilizing a combination of detection methods when evaluating purification strategies. GFP is a well-characterized model protein, used heavily in educational settings and by researchers with limited protein purification experience, and the data and strategies presented here may aid in development other of HIC-compatible protein purification schemes.

Detergent screening of the human voltage-gated proton channel using fluorescence-detection size-exclusion chromatography.

The human voltage-gated proton channel (Hv1) is a membrane protein consisting of four transmembrane domains and intracellular amino- and carboxy-termini. The protein is activated by membrane depolarization, similar to other voltage-sensitive proteins. However, the Hv1 proton channel lacks a traditional ion pore. The human Hv1 proton channel has been implicated in mediating sperm capacitance, stroke, and most recently as a biomarker/mediator of cancer metastasis. Recently, the three-dimensional structures for homologues of this voltage-gated proton channel were reported. However, it is not clear what artificial environment is needed to facilitate the isolation and purification of the human Hv1 proton channel for structural study. In the present study, we generated a chimeric protein that placed an enhanced green fluorescent protein (EGFP) to the amino-terminus of the human Hv1 proton channel (termed EGFP-Hv1). The chimeric protein was expressed in a baculovirus expression system using Sf9 cells and subjected to detergent screening using fluorescence-detection size-exclusion chromatography. The EGFP-Hv1 proton channel can be solubilized in the zwitterionic detergent Anzergent 3-12 and the nonionic n-dodecyl-β-d-maltoside (DDM) with little protein aggregation and a prominent monomeric protein peak at 48 h postinfection. Furthermore, we demonstrate that the chimeric protein exhibits a monomeric protein peak, which is distinguishable from protein aggregates, at the final size-exclusion chromatography purification step. Taken together, we can conclude that solubilization in DDM will provide a useable final product for further structural characterization of the full-length human Hv1 proton channel.

Protein-Chromophore Interactions in Green Fluorescent Protein (GFP) Studied by Split Protein Reconstitution

We have utilized a split green fluorescent protein (GFP) system to generate a range of semi-synthetic proteins to study the interaction of the autocatalytically-formed chromophore with the protein environment. Using circularly permuted GFP, the alpha helix containing the chromophore has been relocated to the N-terminus and then removed by site-specific proteolysis of an engineered loop region. Following denaturation and size exclusion chromatography purification, the truncated protein and the chromophore-bearing peptide are separated. We previously showed that the truncated protein is capable of re-forming a chromophore upon reconstitution with a synthetic peptide. This work is extended to incorporate unnatural amino acids into the chromophore for studying hydrogen bonding and proton transfer. An additional application we have pursued is to use chromophore "transplantation" in order to examine the spectroscopic effects of mutations that would ordinarily be impossible due to the preclusion of chromophore formation. Arg96, in particular, is an interesting residue because of its major role in defining the electrostatic environment, however, its intolerance to mutation necessitates the use of a split protein and complementation with a chromophore-containing peptide harvested from a non-mutated donor. We use this scheme to investigate the effects that remodeling the electrostatics at this critical position have on the color tuning and quantum yield of GFP.

Size exclusion chromatography as a universal method for the purification of quantum dots bioconjugates

The bioconjugation of fluorescent quantum dots (QDs) and purification of QDs bioconjugates are of vital importance in bioapplications. In this paper, we systematically investigated the purification efficiency of QDs bioconjugates and their stability during separation process by using size exclusion chromatography (SEC) technique, fluorescence spectroscopy, and fluorescence correlation spectroscopy (FCS). In this study, commercial QDs and fluorescein isothiocyanate (FITC) were used as labeling probes, and bovine serum albumin (BSA) and antibody (Erbitux) were used as mode samples. The covalently linkage and the electrostatic interaction were used in bioconjugation procedures. We systematically studied the effects of certain factors such as the scales of column, loading volume, elution buffer, and separation media on the purification efficiency of QDs bioconjugates by a home-built SEC device. And highly pure and monodisperse QDs bioconjugates could be obtained by SEC purification twice under the optimized conditions. Furthermore, we investigated the stability of QDs bioconjugates in different conjugation ways and purification conditions by FCS, and found that the stability of bioconjugates were poor when electrostatic binding mode was used. We also observed that Sephacryl S300 HR (separation range for globular proteins: 1 × 10(4) -1.5 × 10(6) Da) was the best choice for purifying the vast majority of QDs-bioconjugates. Our work described here will be constructive to popularization and applications of QDs in life science.

Synthesis, characterization, and antitumor evaluation of the albumin–SN38 conjugate

7-Ethyl-10-hydroxycamptothecin (SN38), the active metabolite of irinotecan, exerts a 100-fold to 1000-fold higher effect than irinotecan itself against several tumor cell lines. However, the water insolubility of SN38 has prevented its direct use as an antitumor drug in the clinic. To improve the water solubility and antitumor efficacy, SN38 was covalently attached to the only free sulfhydryl at cysteine-34 on the BSA site specifically through a thiol-binding linker to form a prodrug BSA-SN38 conjugate (BSA : SN38=1 : 1). The water solubility of this conjugate was similar to albumin using the current method. Also, SN38 loading in this conjugate became controllable. Size-exclusion chromatography purification and UV characterization of the SDS-PAGE electrophoresis product were carried out. Then, an MTT assay was carried out to test the antitumor effect of this conjugate on five colon cancer cell lines in vitro. The 72 h IC50 values of the BSA-SN38 conjugate ranged from 1.5 to 6.1 μmol/l. A colorectal peritoneal carcinomatosis model in mice was established to determine the intraperitoneal chemotherapy effect of the BSA-SN38 conjugate. The BSA-SN38 conjugate at an SN38 equivalent dose of 10 mg/kg/day was administrated every 4 days. Eighteen days after manipulation, the mice were euthanized and the tumors in the abdominal cavity were collected and weighed. Tumors in the BSA-SN38 conjugate treatment group (m=0.21 ± 0.15 g) were found to be significantly (P=5) lighter than those in the NS control group (m=4.74±0.73 g). The results indicated that this water-soluble BSA-SN38 conjugate exerted a strong antitumor effect on colorectal carcinoma.

Specific Soluble Oligomers of Amyloid-β Peptide Undergo Replication and Form Non-fibrillar Aggregates in Interfacial Environments

Aggregates of amyloid-β (Aβ) peptides have been implicated in the etiology of Alzheimer disease. Among the different forms of Aβ aggregates, low molecular weight species ranging between ~2- and 50-mers, also called "soluble oligomers," have emerged as the species responsible for early synaptic dysfunction and neuronal loss. Emerging evidence suggests that the neurotoxic oligomers need not be formed along the obligatory nucleation-dependant fibril formation pathway. In our earlier work, we reported the isolation of one such "off-pathway" 12-18-mer species of Aβ42 generated from fatty acids called large fatty acid-derived oligomers (LFAOs) (Kumar, A., Bullard, R. L., Patel, P., Paslay, L. C., Singh, D., Bienkiewicz, E. A., Morgan, S. E., and Rangachari, V. (2011) PLoS One 6, e18759). Here, we report the physiochemical aspects of LFAO-monomer interactions as well as LFAO-LFAO associations in the presence of interfaces. We discovered that LFAOs are a replicating strain of oligomers that recruit Aβ42 monomers and quantitatively convert them into LFAO assemblies at the expense of fibrils, a mechanism similar to prion propagation. We also found that in the presence of hexane-buffer or chloroform-buffer interfaces LFAOs are able to associate with themselves to form larger but non-fibrillar aggregates. These results further support the hypothesis that low molecular weight oligomers can be generated via non-fibril formation pathways. Furthermore, the unique replicating property of off-pathway oligomers may hold profound significance for Alzheimer disease pathology.

Origin Single-stranded DNA Releases Sld3 Protein from the Mcm2–7 Complex, Allowing the GINS Tetramer to Bind the Mcm2–7 Complex

The replication fork helicase in eukaryotic cells is comprised of Cdc45, Mcm2-7, and GINS (CMG complex). In budding yeast, Sld3, Sld2, and Dpb11 are required for the initiation of DNA replication, but Sld3 and Dpb11 do not travel with the replication fork. Sld3 and Cdc45 bind to early replication origins during the G(1) phase of the cell cycle, whereas Sld2, GINS, polymerase ε, and Dpb11 form a transient preloading complex that associates with origins during S phase. We show here that Sld3 binds tightly to origin single-stranded DNA (ssDNA). CDK-phosphorylated Sld3 binds to origin ssDNA with similar high affinity. Origin ssDNA does not disrupt the interaction between Sld3 and Dpb11, and origin ssDNA does not disrupt the interaction between Sld3 and Cdc45. However, origin ssDNA substantially disrupts the interaction between Sld3 and Mcm2-7. GINS and Sld3 compete with one another for binding to Mcm2-7. However, in a mixture of Sld3, GINS, and Mcm2-7, origin ssDNA inhibits the interaction between Sld3 and Mcm2-7, whereas origin ssDNA promotes the association between GINS and Mcm2-7. We also show that origin single-stranded DNA promotes the formation of the CMG complex. We conclude that origin single-stranded DNA releases Sld3 from Mcm2-7, allowing GINS to bind Mcm2-7.

Structure of Apolipoprotein A-I N Terminus on Nascent High Density Lipoproteins

Apolipoprotein A-I (apoA-I) is the major protein component of high density lipoproteins (HDL) and a critical element of cholesterol metabolism. To better elucidate the role of the apoA-I structure-function in cholesterol metabolism, the conformation of the apoA-I N terminus (residues 6-98) on nascent HDL was examined by electron paramagnetic resonance (EPR) spectroscopic analysis. A series of 93 apoA-I variants bearing single nitroxide spin label at positions 6-98 was reconstituted onto 9.6-nm HDL particles (rHDL). These particles were subjected to EPR spectral analysis, measuring regional flexibility and side chain solvent accessibility. Secondary structure was elucidated from side-chain mobility and molecular accessibility, wherein two major α-helical domains were localized to residues 6-34 and 50-98. We identified an unstructured segment (residues 35-39) and a β-strand (residues 40-49) between the two helices. Residues 14, 19, 34, 37, 41, and 58 were examined by EPR on 7.8, 8.4, and 9.6 nm rHDL to assess the effect of particle size on the N-terminal structure. Residues 14, 19, and 58 showed no significant rHDL size-dependent spectral or accessibility differences, whereas residues 34, 37, and 41 displayed moderate spectral changes along with substantial rHDL size-dependent differences in molecular accessibility. We have elucidated the secondary structure of the N-terminal domain of apoA-I on 9.6 nm rHDL (residues 6-98) and identified residues in this region that are affected by particle size. We conclude that the inter-helical segment (residues 35-49) plays a role in the adaptation of apoA-I to the particle size of HDL.