Silver-stained SDS-PAGE Research Articles

Multifunctional hydrophobin-like protein (HFB-NJ1): A versatile solution for wastewater treatment

As wastewater contains a variety of contaminating bacteria and oily residues, there is an urgent need for environmentally safe bactericidal agents and surfactants which can be applied for wastewater treatment. The present study emphasizes on the potential of hydrophobin-like protein (HFB-NJ1) extracted from sporulating mycelia of Aspergillus sp. NJ1 for wastewater treatment. The purified HFB-NJ1, depicted the presence of one single protein band of molecular size approximately 11–12 kDa on silver-stained SDS-PAGE gel. HFB-NJ1 also presented properties such as surface modification of glass and stable emulsification of sunflower oil. HFB-NJ1 depicted exceptional antibacterial activity against bacterial pathogens such as Bacillus subtilis and Pseudomonas aeruginosa at low MIC of 0.5 μg/mL and 0.75 μg/mL respectively. Additionally, HFB-NJ1 depicted enhanced emulsification of various vegetable and petroleum-based oils (E24 > 80%). HFB-NJ1 effectively reduced gold ions, producing nanospheres with a size of 15.33 nm – a recognized antimicrobial agent. This study underscores the multifunctional attributes of HFB-NJ1, highlighting its efficacy in removing pathogenic bacteria, emulsifying organic compounds from wastewater, and demonstrating a reduction ability for nanoparticle synthesis.

#3230 CHARACTERISTICS OF PROTEIN LOSS WITH MEDIUM CUT-OFF MEMBRANES COMPARED TO HIGH FLUX MEMBRANES

Abstract Background and Aims Medium cut-off membranes (MCO) have been introduced with the claim that larger pores enhance the removal of middle-sized uremic toxins. To date, no adequately powered randomized controlled trial has demonstrated that this membrane characteristic translates into improved mortality. A well-described consequence of larger pore sizes is a higher dialytic albumin loss [1]. In addition, other proteins could also be lost with MCO, including ones that are beneficial or even essential, e.g., to patients’ nutritional status. To better understand the spectrum of proteins cleared with MCO, we set up an ex vivo HD simulation system and compared protein loss characteristics between MCO and high-flux (HF) dialyzers. Method Over the course of an hour, a single reservoir of 2.5 liters EDTA human plasma was ultrafiltered using two Fresenius 2008T machines. One machine served the MCO dialyzer (Theranova 400), the other the HF dialyzer (Fresenius Optiflux F180NR). Ultrafiltrate (UF) was returned to the plasma reservoir (Figure 1A). Blood flow was 400ml/min, dialysate flow was zero, ultrafiltration rate was 13ml/min. UF samples were taken at ∼10 min intervals. Upon experiment completion, residual filtrate was collected from the dialysate compartment. Samples were stored at −80°C until analysis with Bradford protein assay, silver-stained SDS-Page, and Olink Proteomics (Watertown, MA). Selected samples underwent 3K MWCO protein concentration. Results Figure 1B shows the UF protein concentration at several time points. Total protein loss (in mg) during the one-hour simulation was 18 (HF) and 239 (MCO) in the UF, and 10 and 359 in the residual filtrate, respectively. SDS-PAGE protein gel revealed that HF filtrate contained less amount of protein than MCO filtrate when equal filtrate volumes were loaded (Figure 1C, lane 3 vs 2, lane 6 vs 5). Smaller proteins were more abundant in HF vs MCO filtrate when equal amounts of protein were loaded (lane 4 vs 2, lane 7 vs 5). UF from both membranes contained less proteins >100 kD than plasma (lane 1). Finally, UF (collected at 20 and 60 min) and residual filtrate were concentrated to ∼1mg/ml and analyzed using targeted biomarker analysis with Olink® Target 96 Organ Damage panel. Ten of the 92 panel proteins were above the limit of detection (LOD; Table 1). The MCO-to-HF protein concentration ratio in equal filtrate volumes ranged from 1.7 to 13.1. Conclusion Our results show a higher amount of protein loss with MCO compared to HF. The HF filtrate protein distribution is skewed towards lower molecular weights compared to MCO filtrate. All 10 proteins identified were more abundant in MCO filtrate compared to HF filtrate. Further identification and quantification of protein loss during hemodialysis will improve our understanding of the clinical impact of different dialysis membranes.

ADP/ATP translocase 1 protects against an \u03b1-synuclein-associated neuronal cell damage in Parkinson\u2019s disease model

BackgroundADP/ATP translocase 1 (ANT1) is involved in the exchange of cytosolic ADP and mitochondrial ATP, and its defection plays an important role in mitochondrial pathogenesis. To reveal an etiological implication of ANT1 for Parkinson’s disease (PD), a neurodegenerative disorder, a mouse model treated with 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine and neuroblastoma cell model induced by 1-methyl-4-pehny1-pyridine were utilized in this study.ResultsThe tissue-specific abundance in ANT1 in mouse brains was accessed using the analysis of Western blot and immunohistochemistry. Down-regulated soluble ANT1 was found to be correlated with PD, and ANT1 was associated with PD pathogenesis via forming protein aggregates with α-synuclein. This finding was confirmed at cellular level using neuroblastoma cell models. ANT1 supplement in neuronal cells revealed the protective roles of ANT1 against cytotoxicity caused by MPP+. Protein interaction assay, coupled with the analysis of LC-MS/MS, silver-stained SDS-PAGE and Western blot against anti-ANT1 antibody respectively, illustrated the interaction of ANT1 with α-synuclein using the expressed α-synuclein as a bite. Additionally, a significant increasing ROSs was detected in the MPP+-treated cells.ConclusionsThis study indicated that ANT1 was a potentially causative factor of PD, and led to neuropathogenic injury via promoting the formation of protein aggregates with α-synuclein. This investigation potentially promotes an innovative understanding of ANT1 on the etiology of PD and provides valuable information on developing potential drug targets in PD treatment or reliable biomarkers in PD prognostication.

Characterization of Novel Protein Component as Marker for Floral Origin of Jujube (Ziziphus jujuba Mill.) Honey.

Jujube (Ziziphus jujuba Mill.) honey, one of the most valuable honey varieties from China with unique characteristics, is vulnerable to being the target of adulteration and deliberate mislabeling of botanical origin. This study investigated the typical protein component of jujube honey to authenticate the floral source by SDS-PAGE analysis combined with LC-MS/MS identification, and its stability to heating was also evaluated. One band and two adjacent but independent bands, both with molecular weights of ∼19 kDa, were notably observed in Coomassie brilliant blue- and silver-stained SDS-PAGE gels, respectively, for jujube honey from different geographic origins, whereas that was not present for the other five botanical honey varieties, suggesting this protein component was suitable as a marker for jujube honey. LC-MS/MS identification revealed that it was constituted by one Z. jujuba-derived protein (gene number:Zj.jz016003045) and two A. mellifera-derived proteins (an uncharacterized protein with accession number tr|A0A088AC16 and a cleavage fragment from major royal jelly protein-1), and the existence of plant-derived protein was attributed to the special neutral pH of jujube honey. Additionally, these protein markers exhibited good stability to heating below 85 °C/30 min. This study provided a simple method to characterize jujube honey and first identified a protein indicator to determine the botanical origin of honey.

Highly Sensitive SDS Capillary Gel Electrophoresis with Sample Stacking Requiring Only Nanograms of Adeno-Associated Virus Capsid Proteins.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) has been the method of choice in the past decades for size-based protein analysis. However, in general it requires the protein concentration in mg/mL level and thus is not practical for trace level protein analysis, not to mention the lengthy labor-intensive procedures. The SDS capillary gel electrophoresis (SDS CGE) method reported herein requires only nanogram-sized proteins loaded onto the autosampler. A sample stacking technique (e.g., head-column field-amplified sample stacking (HC FASS)) was employed, providing three orders of magnitude sensitivity enhancement compared to conventional SDS CGE. This method has been used routinely in purity analysis and characterization of adeno-associated virus (AAV) intermediates and finished gene therapeutics of AAV vectors. The sensitivity achieved is comparable to the currently most sensitive size-based protein assay silver-stained SDS PAGE. The highly sensitive sample stacking SDS CGE can be used for other types of proteins as well.

Profile proteins/peptides composition in zein excipient by silver staining SDS-PAGE and LC-MS/MS

Profile proteins/peptides composition in zein excipient by silver staining SDS-PAGE and LC-MS/MS

Protocol for the Analysis of Laser Capture Microdissected Fresh-Frozen Tissue Homogenates by Silver-Stained 1D SDS-PAGE.

The heterogeneity present in solid tumors adds significant difficulty to scientific analysis and improved understanding. Fundamentally, solid tumor formation consists of cancer cells proper along with stromal elements. The burgeoning malignant process is dependent upon modified stromal elements. Collectively, the stroma forms an essential microenvironment, which is indispensable for the survival and growth of the malignant neoplasm. This cellular heterogeneity makes molecular profiling of solid tumors via mass spectrometry (MS)-based proteomics a daunting task. Laser capture microdissection (LCM) is commonly used to obtain distinct histological cell types (e.g., tumor parenchymal cells, stromal cells) from tumor tissue and attempt to address the tumor heterogeneity interference with downstream liquid chromatography (LC) MS analysis. To provide optimal LC-MS analysis of micro-scale and/or nano-scale tissue sections, we modified and optimized a silver-stained one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) protocol for the LC-MS analysis of LCM-procured fresh-frozen tissue specimens. Presented is a detailed in-gel digestion protocol adjusted specifically to maximize the proteome coverage of amount-limited LCM samples, and facilitate in-depth molecular profiling. Following LCM, targeted tissue sections are further fractionated using silver-stained 1D-SDS-PAGE to resolve and visualize tissue proteins prior to in-gel digestion and subsequent LC-MS analysis.

Preparative SDS PAGE as an Alternative to His-Tag Purification of Recombinant Amelogenin.

Recombinant protein technology provides an invaluable source of proteins for use in structure-function studies, as immunogens, and in the development of therapeutics. Recombinant proteins are typically engineered with “tags” that allow the protein to be purified from crude host cell extracts using affinity based chromatography techniques. Amelogenin is the principal component of the developing enamel matrix and a frequent focus for biomineralization researchers. Several groups have reported the successful production of recombinant amelogenins but the production of recombinant amelogenin free of any tags, and at single band purity on silver stained SDS PAGE is technically challenging. This is important, as rigorous structure-function research frequently demands a high degree of protein purity and fidelity of protein sequence. Our aim was to generate His-tagged recombinant amelogenin at single band purity on silver stained SDS PAGE for use in functionality studies after His-tag cleavage. An acetic acid extraction technique (previously reported to produce recombinant amelogenin at 95% purity directly from E. coli) followed by repeated rounds of nickel column affinity chromatography, failed to generate recombinant amelogenin at single band purity. This was because following an initial round of nickel column affinity chromatography, subsequent cleavage of the His-tag was not 100% efficient. A second round of nickel column affinity chromatography, used in attempts to separate the cleaved His-tag free recombinant from uncleaved His-tagged contaminants, was still unsatisfactory as cleaved recombinant amelogenin exhibited significant affinity for the nickel column. To solve this problem, we used preparative SDS PAGE to successfully purify cleaved recombinant amelogenins to single band purity on silver stained SDS PAGE. The resolving power of preparative SDS PAGE was such that His-tag based purification of recombinant amelogenin becomes redundant. We suggest that acetic acid extraction of recombinant amelogenin and subsequent purification using preparative SDS PAGE provides a simple route to highly purified His-tag free amelogenin for use in structure-function experiments and beyond.

Sample Stacking Provides Three Orders of Magnitude Sensitivity Enhancement in SDS Capillary Gel Electrophoresis of Adeno-Associated Virus Capsid Proteins.

Size-based protein analysis utilizing only 25 ng of total proteins has been realized by sodium dodecyl sulfate capillary gel electrophoresis (SDS CGE) with head-column field-amplified sample stacking as an online sample preconcentration technique. This method has been used as a replacement of SDS-PAGE for purity analysis of adeno-associated virus (AAV) therapeutic products of different serotypes and transgenes. A limit of detection of 0.2 ng/mL (3.3 pM) capsid proteins was achieved with convenient UV absorbance detection at 214 nm, equivalent to 20 pg of protein (330 attomole) loaded in the autosampler vial. For purity analysis, only 25 ng of total AAV capsid proteins (4.3 femtomole virus particles) were loaded to the autosampler vial. The sensitivity is comparable to silver-stained SDS-PAGE. The RSD of purity measurement was 0.0-0.8%, comparable to conventional SDS CGE utilizing 0.1-0.5 mg proteins. The new method provided 3 orders of magnitude sensitivity enhancement as compared to conventional SDS CGE. It shares all the advantages of conventional SDS CGE (labor-saving, easy automation, and convenient quantitation) and also the high sensitivity of silver stained SDS-PAGE. The sample stacking SDS CGE technique can be adopted for size-based analysis of other types of proteins. It is especially useful when protein quantity or concentration is not sufficient for regular SDS CGE or SDS-PAGE assay.

Two Glycosyltransferase Genes of Haemophilus parasuis SC096 Implicated in Lipooligosaccharide Biosynthesis, Serum Resistance, Adherence, and Invasion

Haemophilus parasuis is a common opportunistic pathogen known for its ability to colonize healthy piglets and causes Glässer's disease. The lipooligosaccharide (LOS) of H. parasuis is a potential virulence-associated factor. In this study, two putative glycosyltransferases that might be involved in LOS synthesis in H. parasuis SC096 were identified (lgtB and lex-1). Mutants were constructed to investigate the roles of the lgtB and lex-1 genes. The LOS from the ΔlgtB or Δlex-1 mutant showed truncated structure on silver-stained SDS-PAGE gel compared to the wild-type strain. The ΔlgtB and Δlex-1 mutants were significantly more sensitive to 50% porcine serum, displaying 15.0 and 54.46% survival rates, respectively. Complementation of the lex-1 mutant restored the serum-resistant phenotype. Additionally, the ΔlgtB and Δlex-1 strains showed impaired ability to adhere to and invade porcine kidney epithelial cells (PK-15). The above results suggested that the lgtB and lex-1 genes of the H. parasuis SC096 strain participated in LOS synthesis and were involved in serum resistance, adhesion and invasion.

Molecular Reference Materials for Standardization of Allergen Measurements

Allergen measurements are widely used for determination of the potency of allergy vaccines, for environmental exposure assessments, and for validation of IgE molecular diagnostics. With the exception of Bet v 1 and Phl p 5a, few purified allergen standards have been developed. The aim of this study was to produce purified allergen standards, with defined specifications, that could serve as Molecular Reference Materials (MRM) for use in allergy diagnostics, enzyme- or fluorescent immunoassays, and multiplex arrays. Natural and recombinant food, dust mite, pollen and animal allergens (n = 11) were purified by affinity and/or gel-filtration chromatography and analyzed by SDS-PAGE, LC-MS/MS, ELISA, and FEIA or chimeric IgE ELISA. Natural allergens from peanut, dust mite and animal were lyophilized using different buffer conditions. Real time stability data are collected from frozen liquid allergens and lyophilized allergens. Allergen purity, assessed by LC-MS/MS after Trypsin digest and /or silver-stained SDS-PAGE, was >95%. Real time stability tests of frozen liquid allergens (up to 24 months) and frozen lyophilized allergens, showed comparable potency in allergen-specific ELISA and no signs of degradation on SDS-PAGE. Monoclonal antibody ELISA and IgE reactivity of recombinant allergens showed excellent correlations with natural allergens. Optimized, ISO 9001 compliant, bioprocessing pathways have been established to yield pure, immune-reactive natural and recombinant allergens. These MRM are stable and will improve the standardization of allergy diagnostics and vaccines used for immunotherapy.

Deletion of the rfaE gene in Haemophilus parasuis SC096 strain attenuates serum resistance, adhesion and invasion

In Haemophilus parasuis, the lipooligosaccharide (LOS) has been identified as an important virulence factor. The rfa gene cluster encodes enzymes for LOS core biosynthesis. In order to investigate the role of the rfaE gene, we generated an rfaE deficient mutant (ΔrfaE) of a H. parasuis SC096 by a natural transformation method. The purified preparation of LOS from the ΔrfaE mutant strain showed truncated LOS structure on silver-stained SDS-PAGE. Compared to the wild-type SC096 strain, the generation time of ΔrfaE mutant strain was significantly extended from 59 min to 69 min. The ΔrfaE mutant strain caused an approximately 30-fold reductions in survival rate in 50% sera and 36-fold reductions in survival rate in 90% sera, respectively (p < 0.001). In adhesion and invasion assays, the ΔrfaE mutant strain had 10-fold less efficient adherence and 12-fold reductions in invasion of the porcine umbilicus vein endothelial cells (PUVEC) and porcine kidney epithelial cells (PK-15), respectively (p < 0.001). However, the complemented strain could restore the above phenotypes. Hence, the above results suggested that the rfaE gene participated in the pathogenicity of H. parasuis SC096 strain.

Large-Scale Purification, Characterization, and Spore Outgrowth Inhibitory Effect of Thurincin H, a Bacteriocin Produced by Bacillus thuringiensis SF361.

Large-scale purification of the highly hydrophobic bacteriocin thurincin H was accomplished via a novel and simple two-step method: ammonia sulfate precipitation and C18 solid-phase extraction. The inhibition spectrum and stability of thurincin H as well as its antagonistic activity against Bacillus cereus F4552 spores were further characterized. In the purification method, secreted proteins contained in the supernatant of a 40 h incubated culture of B. thuringiensis SF361 were precipitated by 68 % ammonia sulfate and purified by reverse-phase chromatography, with a yield of 18.53 mg/l of pure thurincin H. Silver-stained SDS-PAGE, high-performance liquid chromatography, and liquid chromatography-mass spectrometry confirmed the high purity of the prepared sample. Thurincin H exhibited a broad antimicrobial activity against 22 tested bacterial strains among six different genera including Bacillus, Carnobacterium, Geobacillus, Enterococcus, Listeria, and Staphylococcus. There was no detectable activity against any of the selected yeast or fungi. The bacteriocin activity was stable for 30 min at 50 °C and decreased to undetectable levels within 10 min at temperatures above 80 °C. Thurincin H is also stable from pH 2-7 for at least 24 h at room temperature. Thurincin H is germicidal against B. cereus spores in brain heart infusion broth, but not in Tris-NaCl buffer. The efficient purification method enables the large-scale production of pure thurincin H. The broad inhibitory spectrum of this bacteriocin may be of interest as a potential natural biopreservative in the food industry, particularly in post-processed and ready-to-eat food.

Purification of yeast alcohol dehydrogenase by using immobilized metal affinity cryogels

In this study, poly(2-hydroxyethyl methacrylate–glycidylmethacrylate) [poly(HEMA–GMA)] cryogels were prepared by radical cryocopolymerization of HEMA with GMA as a functional comonomer and N,N′-methylene-bisacrylamide (MBAAm) as a crosslinker. Iminodiacetic acid (IDA) functional groups were attached via ring opening of the epoxy group on the poly(HEMA–GMA) cryogels and then Zn(II) ions were chelated with these structures. Characterization of cryogels was performed by FTIR, SEM, EDX and swelling studies. These cryogels have interconnected pores of 30–50μm size. The equilibrium swelling degree of Zn(II) chelated poly(HEMA–GMA)-IDA cryogels was approximately 600%. Zn(II) chelated poly(HEMA–GMA)-IDA cryogels were used in the adsorption of alcohol dehydrogenase from aqueous solutions and adsorption was performed in continuous system. The effects of pH, alcohol dehydrogenase concentration, temperature, and flow rate on adsorption were investigated. The maximum amount of alcohol dehydrogenase adsorption was determined to be 9.94mg/g cryogel at 1.0mg/mL alcohol dehydrogenase concentration and in acetate buffer at pH5.0 with a flow rate of 0.5mL/min. Desorption of adsorbed alcohol dehydrogenase was carried out by using 1.0M NaCI at pH8.0 phosphate buffer and desorption yield was found to be 93.5%. Additionally, these cryogels were used for purification of alcohol dehydrogenase from yeast with a single-step. The purity of desorbed alcohol dehydrogenase was shown by silver-stained SDS–PAGE. This purification process can successfully be used for the purification of alcohol dehydrogenase from unclarified yeast homogenates and this work is the first report about the usage of the cryogels for purification of alcohol dehydrogenase.

Comparative Binding of Disulfide-Bridged PEG-Fabs

Protein PEGylation is the most clinically validated method to improve the efficacy of protein-based medicines. Antibody fragments such as Fabs display rapid clearance from blood circulation and therefore are good candidates for PEGylation. We have developed PEG-bis-sulfone reagents 1 that can selectively alkylate both sulfurs derived from a native disulfide. Using PEG-bis-sulfone reagents 1, conjugation of PEG specifically targets the disulfide distal to the binding region of the Fab (Scheme 2 ). PEG-bis-sulfone reagents 1 (10-40 kDa) were used to generate the corresponding PEG-mono-sulfones 2 that underwent essentially quantitative conjugation to give the PEG-Fab product 4. Four Fabs were PEGylated: Fab(beva), Fab'(beva), Fab(rani), and Fab(trast). Proteolytic digestion of bevacizumab with papain gave Fab(beva), while digestion of bevacizumab with IdeS gave F(ab')(2-beva), which after reaction with DTT and PEG-mono-sulfone 2 gave PEG(2)-Fab'(beva). Ranibizumab, which is a clinically used Fab, was directly PEGylated to give PEG-Fab(rani). Trastuzumab was proteolytically digested with papain, and its corresponding Fab was PEGylated to give PEG-Fab(trast). Purification of the PEGylated Fabs was accomplished by a single ion exchange chromatography step to give pure PEG-Fab products as determined by silver-stained SDS-PAGE. No loss of PEG was detected post conjugation. A comparative binding study by SPR using Biacore with low ligand immobilization density was conducted using (i) VEGF(165) for the bevacizumab and ranibizumab derived products or (ii) HER2 for the trastuzumab derived products. VEGF(165) is a dimeric ligand with two binding sites for bevacizumab. HER2 has one domain for the binding of trastuzumab. Binding studies with PEG-Fab(beva) indicated that the apparent affinity was 2-fold less compared to the unPEGylated Fab(beva). Binding properties of the PEG-Fab(beva) products appeared to be independent of PEG molecular weight. Site-specific conjugation of two PEG molecules gave PEG(2×20)-Fab'(beva), whose apparent binding affinity was similar to that observed for PEG-Fab(beva) derivatives. The k(d) values were similar to those of the unPEGylated Fab(beva); hence, once bound, PEG-Fab(beva) remained bound to the same degree as Fab(beva). Biacore analysis indicated that both Fab(rani) and PEG(20)-Fab(rani) did not dissociate from the immobilized VEGF at 25 °C, but ELISA using immobilized VEGF showed 2-fold less apparent binding affinity for PEG(20)-Fab(rani) compared to the unPEGylated Fab(rani). Additionally, the apparent binding affinities for trastuzumab and Fab(trast) were comparable by both Biacore and ELISA. Biacore results suggested that trastuzumab had a slower association rate compared to Fab(trast); however, both molecules displayed the same apparent binding affinity. This could have been due to enhanced rebinding effects of trastuzumab, as it is a bivalent molecule. Analogous to PEG-Fab(beva) products, PEG(20)-Fab(trast) displayed 2-fold lower binding compared to Fab(trast) when evaluated by ELISA. The variations in the apparent affinity for the PEGylated Fab variants were all related to the differences in the association rates (k(a)) rather than the dissociation rates (k(d)). We have shown that (i) Fabs are well-matched for site-specific PEGylation with our bis-alkylation PEG reagents, (ii) PEGylated Fabs display only a 2-fold reduction in apparent affinity without any change in the dissociation rate, and (iii) the apparent binding rates and affinities remain constant as the PEG molecular weight is varied.

A contaminant trypsin-like activity from the timothy grass pollen is responsible for the conflicting enzymatic behavior of the major allergen Phl p 1

We intend to solve whether or not Phl p 1 can be regarded as a protease. A group reported that Phl p 1 has papain-like properties and later on, that this allergen resembles cathepsin B, while another one demonstrated that Phl p 1 lacks proteinase activity and suggested that the measured activity may rise either from a recombinant Phl p 1 contaminant or as a result of an incompletely purified natural allergen. A third group reported Phl p 1 to act by a non-proteolytic activity mechanism. We report the purification of the natural Phl p 1 by means of hydrophobic interaction, gel filtration and STI-Sepharose affinity chromatographies. The Phl p 1 purity was assessed by silver-stained SDS-PAGE and by ‘in-gel’ and ‘gel-free’ approaches associated to mass spectrometry analyses. The proteolytic activity was measured using Boc–Gln–Ala–Arg–AMC and Z-Phe–Arg–AMC as substrates. While amidolytic activity could be measured with Phl p 1 after rechromatography on gel filtration, it however completely disappeared after chromatography on STI-Sepharose. The contaminant activity co-eluting with Phl p 1 was not affected by cysteine proteases inhibitors and other thiol-blocking agents, by metalloproteases inhibitors and by aspartic proteases inhibitors. However, it was completely inhibited by low molecular weight and proteinaceous serine proteases inhibitors. TLCK, but not TPCK, inhibited the contaminant activity, showing a trypsin-like behavior. The pH and temperature optimum were 8.0 and 37°C, respectively. These data indicated that Phl p 1 is not a protease. The contaminant trypsin-like activity should be considered when Phl p 1 allergenicity is emphasized.

Analysis of Oligomer Assembly for the GluA2 Amino Terminal Domain

For GluA2, an ionotropic glutamate receptor that mediates excitatory synaptic transmission at CNS synapses, the reported monomer-dimer Kd of the amino-terminal domain (ATD) varies widely, ranging from 1.8 nM to 4 μM.1-3 To investigate causes of discrepancies in the literature, several protein constructs, diverse in glycosylation and overall size, were created and the dimer Kd analyzed by analytical ultracentrifugation with absorbance and interference optics (AUC). Sedimentation velocity (SV) Kds varied from 2 to 11 nM, with size and glycosylation having little effect. At the highest protein concentration examined (33 μM) we did not detect formation of tetramers or larger oligomer species. Steady state fluorescence anisotropy titrations for DyLight405 labelled protein yielded similar results. However, sedimentation equilibrium analysis yielded dissociation constants of 160 to 280 nM. Temperature and pressure variations did not explain this difference. Small amounts of degradation over the several day sedimentation equilibrium protocol was revealed by silver-stained SDS-PAGE and likely underlies the different Kds for sedimentation velocity and sedimentation equilibrium experiments. Our results confirm that the GluA2 ATD forms nM affinity dimers3. The spread of values measured by SV highlights the difficulty in making accurate measurements at nanomolar protein concentrations, due to dimer-deficient monomers produced by proteolysis or extremely small signal to noise ratios. The similar variance observed in prior work with fluorescence detection AUC suggests that S/N limitations are not the major cause of Kd variations. 1. Jin R, Singh SK, Gu S, Furukawa H, Sobolevsky AI, Zhou J, Jin Y, Gouaux E (2009). EMBO J 28:1812-1823. 2. Clayton A, Siebold C, Gilbert RJ, Sutton GC, Harlos K, McIlhinney RA, Jones EY, Aricescu AR (2009) J Mol Biol 392:1125-1132. 3. Rossmann M, Sukumaran M, Penn AC, Veprintsev DB, Babu MM, Greger IH (2011) EMBO J 30:959-971.

A proteomic approach to analyzing responses of Arabidopsis thaliana root cells to different gravitational conditions using an agravitropic mutant, pin2 and its wild type

BackgroundRoot gravitropsim has been proposed to require the coordinated, redistribution of the plant signaling molecule auxin within the root meristem, but the underlying molecular mechanisms are still unknown. PIN proteins are membrane transporters that mediate the efflux of auxin from cells. The PIN2 is important for the basipetal transport of auxin in roots and plays a critical role in the transmission of gravity signals perceived in the root cap to the root elongation zone. The loss of function pin2 mutant exhibits a gravity-insensitive root growth phenotype. By comparing the proteomes of wild type and the pin2 mutant root tips under different gravitational conditions, we hope to identify proteins involved in the gravity-related signal transduction.ResultsTo identify novel proteins involved in the gravity signal transduction pathway we have carried out a comparative proteomic analysis of Arabidopsis pin2 mutant and wild type (WT) roots subjected to different gravitational conditions. These conditions included horizontal (H) and vertical (V) clinorotation, hypergravity (G) and the stationary control (S). Analysis of silver-stained two-dimensional SDS-PAGE gels revealed 28 protein spots that showed significant expression changes in altered gravity (H or G) compared to control roots (V and S). Whereas the majority of these proteins exhibited similar expression patterns in WT and pin2 roots, a significant number displayed different patterns of response between WT and pin2 roots. The latter group included 11 protein spots in the H samples and two protein spots in the G samples that exhibited an altered expression exclusively in WT but not in pin2 roots. One of these proteins was identified as annexin2, which was induced in the root cap columella cells under altered gravitational conditions.ConclusionsThe most interesting observation in this study is that distinctly different patterns of protein expression were found in WT and pin2 mutant roots subjected to altered gravity conditions. The data also demonstrate that PIN2 mutation not only affects the basipetal transport of auxin to the elongation zone, but also results in an altered expression of proteins in the root columella.

El papel de los carbohidratos en la antigenicidad de las glicoproteínas del cisticerco de la Taenia solium.

The glycoproteins af 12-28 kD from Taenia solium rnetaoestcdes provide a high specificity and sensitivei for the serological diagnosis of the central nervous systern infection, neurocysticercosis. their widespread use as antigens for routine serlogical assays will require their production in large and reproducible amounts. Prior to deterrnining the ideal strategy to produce these antigens at a large scale, it is important to determine the contribution of the carbohydrates to the antigenicity of these molecules, given the uncertainty of reproducing saccharidic epitopes in recombinant expression systems. In thia study we examined this issue. The chemical oxidation of the carbohydrates of the 12-28 kD glycsproteins with sodium metaperiodate, reduced the antigenicity of the rnolecules to variable extents, with the more notable changes being detected for the 18 and 28 kD antigens. This approach was complemented by purification of the 12, 16 and 18 k5 antigens, followed by the enzymatic deglycosylation af their abundant N-linked oligosaccharides. Silver-stained SDS-PAGE anatysis indicated that the three deglycosylated antigens now migrated as 7 kD products, suggesting a protein backbone with a similar size, but different sxtents of glycosylation. By Western blot, the antigenicity of these antigens was diminished. This was more notable for the 18 kD antigen,which is more heavily glycosylated than the 12 or 16 kD glycoproteins. These data suggest that the antigenicity of the glycoproteins of T: solium is due to a combination of carbohydrate and protein epitopes.

Nanodisc-based Co-immunoprecipitation for Mass Spectrometric Identification of Membrane-interacting Proteins

Proteomic identification of protein interactions with membrane associated molecules in their native membrane environment pose a challenge because of technical problems of membrane handling. We investigate the possibility of employing membrane nanodiscs for harboring the membrane associated molecule to tackle the challenges. Nanodiscs are stable, homogenous pieces of membrane with a discoidal shape. They are stabilized by an encircling amphipatic protein with an engineered epitope tag. In the present study we employ the epitope tag of the nanodiscs for detection and co-immunoprecipitation of interaction partners of the glycolipid ganglioside GM1 harbored by nanodiscs. Highly specific binding activity for nanodisc-GM1 immobilized on sensorchips was observed by surface plasmon resonance in culture media from enterotoxigenic Escherischia coli. To isolate the interaction partner(s) from enterotoxigenic Escherischia coli, GM1-nanodiscs were employed for co-immunoprecipitation. The B subunit of heat labile enterotoxin was identified as a specific interaction partner by mass spectrometry, thus demonstrating that nanodisc technology is useful for highly specific detection and identification of interaction partners to specific lipids embedded in a membrane bilayer.