Non-reducing SDS-PAGE Research Articles

Reinvestigation of Disulfide-bonded Oligomeric Forms of the Unfolded Protein Response Transducer ATF6.

ATF6α is an endoplasmic reticulum (ER)-embedded transcription factor which is rapidly activated by ER stress, and a major regulator of ER chaperone levels in vertebrates. We previously suggested that ATF6α occurs as a monomer, dimer and oligomer in the unstressed ER of Chinese hamster ovary cells due to the presence of two evolutionarily conserved cysteine residues in its luminal region (C467 and C618), and showed that ATF6α is reduced upon ER stress, such that only reduced monomer ATF6α is translocated to the Golgi apparatus for activation by proteolysis. However, mutagenesis analysis (C467A and C618A) revealed that the C618A mutant behaves in an unexpected manner (monomer and oligomer) during non-reducing SDS-PAGE, for reasons which remained unclear. Here, we used human colorectal carcinoma-derived HCT116 cells deficient in ATF6α and its relevant ATF6β, and found that ATF6α dimer and oligomer are both dimers, which we designated C618-dimer and C467-dimer, respectively. We demonstrated that C467-dimer (previously considered an oligomer) behaved bigger than C618-dimer (previously considered a dimer) during non-reducing SDS-PAGE, based on their disulfide-bonded structures. Furthermore, ATF6α monomer physically associates with another ATF6α monomer in the absence of disulfide bonding, which renders two C467 residues in close proximity so that formation of C467-dimer is much easier than that of C618-dimer. In contrast, C618-dimer is more easily reduced upon ER stress. Thus, our analysis revealed that all forms of ATF6α, namely monomer, C618-dimer and C467-dimer, are activated by single reduction of a disulfide bond in response to ER stress, ensuring the rapidity of ATF6α activation.Key words: disulfide-bonded structure, endoplasmic reticulum, membrane-bound transcription factor, non-reducing SDS-PAGE, unfolded protein response.

Isolation of Nile crocodile (Crocodylus niloticus) serum immunoglobulin M and Y (IgM and IgY)

Crocodile immunity has not been fully characterised with more studies on crocodile innate immunity than cell-mediated or humoral immunity. Crocodile immunoglobulin genes have been described but immunoglobulin proteins have not been isolated or studied biochemically. Two large proteins proposed to be crocodile IgM and IgY were isolated and purified from Crocodylus niloticus sera using two different protocols. A 50% (w/v) ammonium sulfate and a 15% (w/v) polyethylene glycol precipitation step was followed by Cibacron blue F3GA affinity- and Sephacryl-S300 gel filtration chromatography. An alternate purification protocol, with only two steps, involved thiophilic affinity- and Sephacryl-S300 gel filtration chromatography. The purified crocodile IgM resolved on reducing SDS-PAGE with an apparent mass of 180 kDa. Purified crocodile IgY resolved at 180 kDa alongside chicken IgY on a non-reducing SDS-PAGE gel, and is deduced to consist of two 66 kDa heavy and two 23 kDa light chains under reducing conditions. The thiophilic/gel filtration two-step protocol gave three-fold higher yields of isolated protein than the four-step precipitation/chromatography protocol. Antibodies against the isolated crocodile IgM and IgY were raised in chickens and affinity purified. The chicken antibodies differentiated between crocodile IgM and IgY and have the potential for use in the diagnosis of crocodile infections. The purified crocodile antibodies can be biochemically characterised and compared to mammalian and avian antibodies to give a better understanding of crocodile humoral immunity.

Combining Non-reducing SDS-PAGE Analysis and Chemical Crosslinking to Detect Multimeric Complexes Stabilized by Disulfide Linkages in Mammalian Cells in Culture.

The structures of many proteins are stabilized through covalent disulfide linkages. In recent work, this bond has also been classified as a post-translational modification. Thus, it is important to be able to study this modification in living cells. A simple method to analyze these cysteine-stabilized multimeric complexes is through a two-step method of non-reducing SDS-PAGE analysis and formaldehyde cross-linking. This two-step method is advantageous as the first step to uncovering multimeric complexes stabilized by disulfide linkages due to its technical ease and low cost of operation. Here, the human bone osteosarcoma cell line U-2 OS is used to illustrate this method by specifically analyzing the nuclear isoform of dUTPase.

Combining Non-reducing SDS-PAGE Analysis and Chemical Crosslinking to Detect Multimeric Complexes Stabilized by Disulfide Linkages in Mammalian Cells in Culture

Combining Non-reducing SDS-PAGE Analysis and Chemical Crosslinking to Detect Multimeric Complexes Stabilized by Disulfide Linkages in Mammalian Cells in Culture

Plant glutathione biosynthesis revisited: redox-mediated activation of glutamylcysteine ligase does not require homo-dimerization.

Plant γ-glutamylcysteine ligase (GCL), catalyzing the first and tightly regulated step of glutathione (GSH) biosynthesis, is redox-activated via formation of an intramolecular disulfide bond. In vitro, redox-activation of recombinant GCL protein causes formation of homo-dimers. Here, we have investigated whether dimerization occurs in vivo and if so whether it contributes to redox-activation. FPLC analysis indicated that recombinant redox-activated WT (wild type) AtGCL dissociates into monomers at concentrations below 10−6 M, i.e. below the endogenous AtGCL concentration in plastids, which was estimated to be in the micromolar range. Thus, dimerization of redox-activated GCL is expected to occur in vivo. To determine the possible impact of dimerization on redox-activation, AtGCL mutants were generated in which salt bridges or hydrophobic interactions at the dimer interface were interrupted. WT AtGCL and mutant proteins were analyzed by non-reducing SDS–PAGE to address their redox state and probed by FPLC for dimerization status. Furthermore, their substrate kinetics (KM, Vmax) were compared. The results indicate that dimer formation is not required for redox-mediated enzyme activation. Also, crystal structure analysis confirmed that dimer formation does not affect binding of GSH as competitive inhibitor. Whether dimerization affects other enzyme properties, e.g. GCL stability in vivo, remains to be investigated.

Formation of Disulfide Bonds in Proinsulin is Dependent on Both the Substrate Chemical Form and the Conditions Used for Purification and Folding

Misfolding of proinsulin (PI), the biosynthetic precursor to insulin, is believed to be the underlying cause of some forms of both type 1 and type 2 diabetes mellitus. It has been proposed that the ER stress response plays a role in recognizing and responding to PI misfolding in the cell. My laboratory is developing an in vitro model system to examine the roles of PI, protein disulfide isomerase (PDI) and the redox co‐factor glutathione in the folding reaction.Both PI and PDI were expressed as N‐terminal poly‐His‐tagged proteins in bacteria. The PI fusion protein was recovered from inclusion bodies and the poly‐His tag was removed by cyanogen bromide cleavage. Liberated PI was recovered by rotary evaporation and subjected to further chemical treatment. PDI was purified from the soluble fraction of bacteria using a combination of IMAC and gel filtration chromatography.Two forms of PI were studied: sulfonated and reduced (PI‐SO4 and PI‐red, respectively). Both versions of PI were subjected to gel filtration chromatography at pH 7.2. PI‐SO4 eluted within the included volume of the column while PI‐red eluted at the void volume, suggesting that the latter version had undergone significant aggregation. Subsequent analysis using non‐reducing SDS‐PAGE indicated that both forms of PI migrated through the gel as monomers. Reaction of PI‐red with iodoacetic acid prior to non‐reducing SDS‐PAGE resulted in a mobility shift, reinforcing the hypothesis that the aggregation event was not due to the formation of incorrect disulfide cross‐links between PI molecules.Folding of both versions of PI (25 μM) was tested in the presence of reduced and oxidized glutathione (GSH/GSSG, 3 mM of each), PDI (5 μM) or a combination of GSH/GSSG and PDI. Previous studies had established that samples of PI‐red taken directly from the reduction reaction (performed in the presence of either 6 M guanidine or 8 M urea); obtained following buffer exchange without chaotropic agents at pH 10.6; or obtained after folding in the presence of GSH/GSSG at pH 10.6; could all be analyzed using RP‐HPLC. However, PI‐red that had been subjected to chromatography between pH 7 and 8 in the absence of chaotropic agents could not be analyzed using this method as no PI‐red peak could be detected and the HPLC column quickly became clogged. As an alternative, native PAGE was investigated as an alternative analytical method. Results from this study indicated that folding of PI‐SO4 at pH 7.2 can occur in the presence of either GSH/GSSG or a combination of GSH/GSSG and PDI, but not in the presence of PDI alone. In contrast, it was found that PI‐red does not enter the native PAGE gels under any of the conditions tested. This suggests that the non‐disulfide bonded, aggregated form of PI‐red could not be properly folded at pH 7.2 using concentrations of PI and GSH/GSSG similar to those that resulted in proper folding at pH 10.6.In conclusion, these results suggest that folding of PI in islet beta cells must take place under tightly controlled conditions and that mimicking these conditions in an in vitro assay will require further understanding of the biochemistry of the PI molecule.Support or Funding InformationSupported by the Creighton University Health Science Strategic Investment FundThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Protein Disulfide Isomerase Inhibits and Reverses Alpha‐Synuclein Fibrillization

Protein disulfide isomerase (PDI) is a ubiquitously expressed member of the thioredoxin family with related but independent oxidoreductase and chaperone properties. As a chaperone, PDI plays a crucial role in preventing the aggregation of misfolded proteins. Notably, PDI is known to inhibit alpha‐synuclein (SYN) fibrillization, a pathological hallmark of Parkinson's disease, but the mechanism by which PDI blocks the formation of SYN fibrils is unknown. Here, we report for the first time that PDI not only prevents SYN aggregation but is able to reverse aggregation both in vitro and in cultured cells. Assays using Thioflavin‐T fluorescence and transmission electron microscopy revealed that PDI is able to break down nascent but not mature fibrils. Furthermore, PDI was able to inhibit and reverse cellular fibrillization in HEK293T cells as assessed by protein‐fragment complementation and non‐reducing SDS‐PAGE. These data suggest a novel disaggregase function for PDI that disrupts the SYN fibrils most commonly found in Parkinson's disease. Current treatments for PD are limited to palliative care, but strategies focused on targeted therapeutic intervention are becoming popular. Thus, our work suggests the disaggregase property of PDI may lend itself to therapeutic development for Parkinson's disease.Support or Funding InformationThis work was supported, in part, by a LIFE at UCF Richard Tucker Gerontology Applied Research Award and a UCF College of Medicine competitive research grant.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of a Recombinant Human Interleukin-12 (rhIL-12) Fragment in Non-Reduced SDS-PAGE.

During the past two decades, recombinant human interleukin-12 (rhIL-12) has emerged as one of the most potent cytokines in mediating antitumor activity in a variety of preclinical models and clinical studies. Purity is a critical quality attribute (CQA) in the quality control system of rhIL-12. In our study, rhIL-12 bulks from manufacturer B showed a different pattern in non-reduced SDS-PAGE compared with size-exclusion chromatography (SEC)-HPLC. A small fragment was only detected in non-reduced SDS-PAGE but not in SEC-HPLC. The results of UPLC/MS and N-terminal sequencing confirmed that the small fragment was a 261–306 amino acid sequence of a p40 subunit of IL-12. The cleavage occurs between Lys260 and Arg261, a basic rich region. With the presence of 0.2% SDS, the small fragment appeared in both native PAGE and in SEC-HPLC, suggesting that it is bound to the remaining part of the IL-12 non-covalently, and is dissociated in a denatured environment. The results of a bioassay showed that the fractured rhIL-12 proteins had deficient biological activity. These findings provide an important reference for the quality control of the production process and the final products of rhIL-12.

Partial purification and characterization of phytase from Aspergillus foetidus MTCC 11682

Phytase is a phosphatase enzyme widely used as feed additive to release inorganic phosphorus from plant phytate and enhance its uptake in monogastric animals. Although engineered fungal phytases are used most, a natural enzyme gives opportunity to understand novel properties, if any. In the current study, a novel fungal strain, Aspergillus foetidus MTCC 11682 was immobilized on poly urethane cubes and used for phytase production, purification and molecular characterization. Phytase produced by this method was partially purified by ammonium sulphate precipitation and Sephacryl S-200HR gel filtration to 23.4-fold (compared to crude extract) with recovery of 13% protein. Electrophoresis analysis revealed that phytase has molecular weight of 90.5 kDa on non-reducing and 129.6 kDa on reducing SDS-PAGE. The purified phytase exhibited a wider pH and temperature stability. Analysis of the cloned sequence showed that the gene has 1176 bp that encodes for a peptide of 391 amino acids of the core catalytic region. It was also found that phytase from A. foetidus has a sequence identity of 99% with the phytase gene of other Aspergillus species at nucleotide level and 100% at protein level in A. niger, A. awamori, A. oryzae. In silico analysis of sequence identified the presence of two consecutive and one non-consecutive intra chain disulfide bonds in the phytase. This probably contributed to the differential migration of phytase on reducing and non-reducing SDS-PAGE. There are predicted 11 O-glycosylation sites and 8 N-glycosylation sites, possibly contributed to an enhanced stability of enzyme produced by this organism. This study opened up a new horizon for exploring the novel properties of phytase for other applications.

TFDP3and DDX53 are Highly Re-Expressed after One Cycle of Azacitidine in Myelodysplastic Syndrome Patients Achieving Complete Response: A Cancer Testis Antigen RNA-Seq Screening

Background and Aim: Azacitidine have shown clinical activity in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), particularly at low, non-cytotoxic doses favoring hypomethylation over cytotoxicity. Cancer/testis antigens (CTAs, encoding for immunogenic proteins which are normally expressed in the testicles and trophoblastic cells of the ovary, have been shown to undergo derepression after the use of demethylating agents in cancer cell line models. We took advantage of the unique model of Aza-treated high risk MDS to in vivocharacterize candidates for immunotherapy following hypometilating therapy.Methods:Targeted RNA-Seq (tRNA-Seq) designed to capture 214 CTA genes was performed in 19 MDS or CMML patients at day 0 and at day +28 after first AZA cycle. In 10 patients the analysis was extended to third and/or four cycles. Sequencing, coverage, mapping and alignment was performed using the Ion Torrent Browser Suite. We used LIMMA package or empirical analysis of digital gene expression data in R (edgeR) to identify differential expressed genes. A homogeneous treatment schedule with AZA (75 mg/m2/day x 7 days) was received by each patient.The response criteria were from IWG 2006, including a bone marrow and cytogenetic reevaluation after 6 cycles. The main candidates selected from de tRNA-seq experiment were characterized at protein level in cell lysate and/or plasma by Western Blot. Primary antibodies used from Thermo Fisher were: ADAM29, DDX53, PRAME, B-TUBULINA, FAM46D, B-ACT, TMPRSS12, MAGEB4, GAPDH, MAGEA1, MAGEA2, TSPY2, Cxorf48, DNAJB8, NY-ES0-1, CT83/Cxorf61 and TFDP3 from Santa Cruz Biotechnology. We used either cell lysate or plasma to study the protein levels in non-reducing SDS-PAGE at different acrylamide concentration depending on the protein size. ImageJ was used to quantify protein levels.Results:The median age of the cohort was 69 (range 48-81 years). The cohort consisted of 16 MDS and 3 CMML patients. MDS Patients were stratified based on IPSS as low or intermediate-1 (n=14) and intermediate-2 or high (n=2) risk groups and CMML patients based on a CPSS of intermediate-2 (n=3). Five patients were classified as complete responders achieving a complete cytogenetic and/or marrow remission (CR). Regarding the tRNA-Seq, on average, we achieved 3.888.308 mapped reads per sample (p25-p75, 1.2x106 - 5.9x106) which represents a sufficient depth for digital gene expression profiling of 214 genes. CTAs re-expressed significantly after one AZA cycle in complete responders were TFDP3 (FC=6,4), ZNF645 (FC=2), MAGEB4 (FC=3,1), MAGEA5 (FC=2), DDX53 (FC=2), VENTPX1 (FC=5,5), TSPY2(FC=3,4) and TSYP3 (FC=3,5). Other potential candidates with significantly re-expression in any responders vs. non responder were:SLCO6A1, ADAM29, DDX53, PRAME, FAM46D, TMPRSS12, MAGEA1, MAGEA2, Cxorf48, DANJB8, NYESO-1 and CT83. In the western-blot experiments, protein expression, longitudinally measured in peripheral blood lysate and/or plasma showed a parallel dynamic to gene expression in the case of TFDP3and DDX53.Conclusions:In this study, targeted RNA-seq is shown as effective and accurate tool to detect weakly expressed transcripts as CTAs. In this work, TFDP3 and DDX53, validated at proteomic level, emerge as most promising candidates for immunotherapy in combination with hypomethylating agents in MDS patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

T = 4 Icosahedral HIV-1 Capsid As an Immunogenic Vector for HIV-1 V3 Loop Epitope Display.

The HIV-1 mature capsid (CA) assumes an amorphous, fullerene conical configuration due to its high flexibility. How native CA self-assembles is still unclear despite having well-defined structures of its pentamer and hexamer building blocks. Here we explored the self-assembly of an engineered capsid protein built through artificial disulfide bonding (CA N21C/A22C) and determined the structure of one fraction of the globular particles. CA N21C/A22C was found to self-assemble into particles in relatively high ionic solutions. These particles contained disulfide-bonding hexamers as determined via non-reducing SDS-PAGE, and exhibited two major components of 57.3 S and 80.5 S in the sedimentation velocity assay. Particles had a globular morphology, approximately 40 nm in diameter, in negative-staining TEM. Through cryo-EM 3-D reconstruction, we determined a novel T = 4 icosahedral structure of CA, comprising 12 pentamers and 30 hexamers at 25 Å resolution. We engineered the HIV-1 V3 loop to the CA particles, and found the resultant particles resembled the morphology of their parental particles in TEM, had a positive reaction with V3-specific neutralizing antibodies, and conferred neutralization immunogenicity in mice. Our results shed light on HIV CA assembly and provide a particulate CA for epitope display.

Gene cloning, expression in E. coli, and in vitro refolding of a lipase from Proteus sp. NH 2-2 and its application for biodiesel production.

To obtain active lipases for biodiesel production by refolding Proteus sp. lipase inclusion bodies expressed in E. coli. A lipase gene lipPN1 was cloned from Proteus sp. NH 2-2 and expressed in E. coli BL21(DE3). Non-reducing SDS-PAGE revealed that recombinant LipPN1(rLipPN1) were prone to form inclusion bodies as disulfide-linked dimers in E. coli. Site-directed mutagenesis confirmed that Cys85 in LipPN1 was involved in the dimer formation. After optimizing the inclusion body refolding conditions, the maximum lipase activity reached 1662 U/L. The refolded rLipPN1 exhibited highest activity toward p-nitrophenyl butyrate at pH 9.0 and 40°C. It could be activated by Ca2+ with moderate tolerance to organic solvents. It could also convert soybean oil into biodiesel at a conversion ratio of 91.5%. Preventing the formation of disulfide bond could enhance the refolding efficiency of rLipPN1 inclusion bodies.

Two-hundred-liter scale fermentation, purification of recombinant human fibroblast growth factor-21, and its anti-diabetic effects on ob/ob mice.

Fibroblast growth factor-21 (FGF-21) is a potential cytokine for type II diabetes mellitus. This study aimed to optimize recombinant human FGF-21 (rhFGF-21) production in Escherichia coli BL21 (DE3) employing high cell density fermentation at a 200-L scale and pilot-scale purification. FGF-21 was eventually expressed in E. coli BL21 (DE3) using human FGF-21 synthetic DNA sequence via the introduction of vector pET-3c; the product is used as seed strain during the fermentation of rhFGF-21. Fermentation of rhFGF-21 was performed in a 30-L and 200-L fermenters. rhFGF-21 was primarily expressed in the form of inclusion bodies after IPTG induction. At the 200-L scale, the bacterial production and expression levels of rhFGF-21 were 38.8 ± 0.6g/L and 30.9 ± 0.7%, respectively. Additionally, the high purification (98%) of rhFGF-21 was tested with HPLC analysis and reducing & non-reducing SDS-PAGE analysis. The final yield of purified rhFGF-21 was 71.1 ± 13.9mg/L. The activity of rhFGF-21 stock solution reached at 68.67 ± 8.74IU/mg. Blood glucose controlling and insulin sensitization were improved with treatment of rhFGF-21 in type II diabetic ob/ob mice. Our results showed that the relatively stable and time-saving pilot-scale production process was successfully established, providing an efficient and cost-effective strategy for large-scale and industrial production of rhFGF-21.

20 - PKM2 S-glutathionylation promotes the reprogramming of macrophage metabolism and activation states

Introduction Pyruvate Kinase Isoform M2 (PKM2) regulates glycolysis in monocytes and macrophages and is induced and S-glutathionylated in response to diet-induced metabolic stress. We hypothesized that PKM2 S-glutathionylation promotes PKM2 tetramer dissociation into glycolytically-inactive dimers and that nuclear translocation of these dimers activates HIF1a-dependent gene expression, inducing both glycolytic enzymes as well and pro-inflammatory proteins, reprogramming macrophages to a hyper-inflammatory phenotype. Methods Bone marrow derived macrophages (BMDM) overexpressing glutaredoxin-1-EGFP (Grx1) or EGFP only were exposed for 36 h to metabolic stress (high glucose plus native human LDL). PKM2 expression and phosphorylation state (Tyr105) were assessed in cell lysates by Western blot analysis. PKM2 S-glutathionylation was quantified using the Biotin-switch assay. PKM2 oligomerization state was measured by non-reducing SDS-PAGE and Western blot analysis. To confirm the role of PKM2 S-glutathionylation on metabolic reprogramming of monocytes in vivo, we conducted lentiviral gene transfer experiments in mice using CD68 promoter-driven Grx1 and EGFP expression. Mice were fed a HFD for 10 week to induce monocyte priming. We purified blood monocytes from EGFPMactg and Grx1Mactg mice and determined by single-cell Western blot PKM2 induction and phosphorylation, GLUT1, 14-3-3z and IL-1b expression. Results PKM2 induction, -glutathionylation, and phosphorylation were all enhanced in metabolically stressed BMDMs but prevented in Grx1-overexpressing BMDMs. The dimer/tetramer ratio was increased in response to metabolic stress as was HIF1a-dependent expression of GLUT1 and IL-1b. Compared to monocytes isolated from HFD-fed mice, monocyte from Grx1Mactg mice showed reduced priming, reduced expression of PKM2, GLUT1 and IL-1b and diminished PKM2 phosphorylation. Conclusion Metabolic stress-induced PKM2 S-glutathionylation promotes PKM2 tetramer dissociation, phosphorylation and subsequent nuclear translocation, which, via HIF1a-dependent expression of glycolytic enzymes and inflammatory cytokines, reprograms monocyte-derived macrophages into a hyper-inflammatory phenotype. This mechanism may contribute to chronic inflammatory diseases associated with metabolic disorders.

Domain unfolding of monoclonal antibody fragments revealed by non-reducing SDS-PAGE

Monoclonal antibodies and derived fragments are used extensively both experimentally and therapeutically. Thorough characterization of such antibodies is necessary and includes assessment of their thermal and storage stabilities. Thus, assessment of the underlying conformational stabilities of the antibodies is also important. We recently documented that non-reducing SDS-PAGE can be used to assess both monoclonal and polyclonal IgG domain thermal unfolding in SDS. Utilizing this same h2E2 anti-cocaine mAb, in this study we generated and analyzed various mAb antibody fragments to delineate the structural domains of the antibody responsible for the observed discrete bands following various heating protocols and analysis by non-reducing SDS-PAGE. Previously, these domain unfolding transitions and gel bands were hypothesized to stem from known mAb structural domains based on the relative thermal stability of those CH2, CH3, and Fab domains in the absence of SDS, as measured by differential scanning calorimetry. In this study, we generated and analyzed F(ab’)2, Fab, and Fc fragments, as well as a mAb consisting of only heavy chains, and examined the thermally induced domain unfolding in each of these fragments by non-reducing SDS-PAGE. The results were interpreted and integrated to generate an improved model of thermal unfolding for the mAb IgG in SDS. These results and the model presented should be generally applicable to many monoclonal and polyclonal antibodies and allow novel comparisons of conformational stabilities between chemically or genetically modified versions of a given antibody. Such modified antibodies and antibody drug conjugates are commonly utilized and important for experimental and therapeutic applications.

Protein Extraction Protocol from Musa sp. Shoots and Roots Tissue for Non-reducing One Dimensional SDS-PAGE Analysis

Background: Protein extraction from plant tissues is a great challenge since they contain low amount of proteins and rich in proteases, secondary metabolites and oxidative enzymes. Besides, dealing with limited amount of starting material would pose another challenge in getting high protein yield. The aim of this study is to determine the best protein extraction protocol for recalcitrant tissues of banana (Musa sp.) specific for non-reducing one dimensional SDS-PAGE analysis. Methods: Three protein extraction protocols were compared which include TCA-acetone, phenol and phosphate buffer saline (PBS) using small amount of starting material. Modifications were done to each protocol to suit non-reducing SDS-PAGE analysis and subsequent downstream processes. Results: Of the three protocols compared, TCA-acetone protocol gave the highest yield and quality of protein extract from only a small amount of starting material based on protein quantification and non-reducing one dimensional SDS-PAGE analysis compared to phenol and phosphate buffer saline (PBS) protocol. Conclusion: Even though various protein extraction protocols have been developed to date, there is no one-size-fits-all when it comes to different types of plant tissues from varieties of plant species. TCA-acetone protocol was found to be most effective in extracting protein from banana shoot and root tissues even for small amount of starting material in absence of reducing agent. Keywords: Protein extraction, recalcitrant tissue, Musa sp., non-reducing SDS-PAGE, TCA-acetone protocol, reducing agents.

Study on the emulsifying stability and interfacial adsorption of pea proteins

In order to expand the natural food emulsifier applications of pea proteins, the emulsifying stability and competitive relationship of interfacial adsorption were investigated. The protein extracted at 90 °C had the highest nitrogen solubility index and surface hydrophobicity. The molecular weight distributions analyzed by high-performance size exclusion chromatography demonstrated that over 60% of the protein had Mw > 500 kDa. The non-reducing SDS-PAGE results showed that the percentage of aggregates were about 37%, and the proportion of proteins were aggregates > vicilin > legumin > convicilin. Increasing the protein concentration from 1.0 to 30 mg/mL increased the emulsifying ability and stability of pea protein stabilized emulsions significantly. At the same time, the concentration of interfacial adsorbed proteins increased. However, the ratio of adsorbed protein to the protein in the initial dispersion (AP%) was decreased significantly from 84% to 21%. When the protein concentration was higher than 10 mg/mL, the interfacial adsorption of pea proteins would reach the saturated adsorption point. The content of aggregates adsorbed onto the interface at low concentration was higher than its proportion in the initial pea protein. With the increase of protein concentration at the interface, the proportion of adsorbed aggregates decreased, while the proportions of vicilin and legumin increased. At saturated adsorption, the contents of proteins on the interface were vicilin > legumin > aggregates > convicilin, and the proportion of aggregates was lower than its proportion in the initial pea protein extraction.

Production and characterization of anti-human IgG F(ab')2 antibody fragment.

In present study an optimized protocol for the separation of antibodies into antigen-binding fragments F(ab')2 using pepsin digestion was investigated. The production of these fragments is a consequential step in the development of medical research, treatment and diagnosis. For production of polyclonal antibody rabbit received antigen in four steps. The rabbit serum at 1/128000 dilution showed high absorbance in reaction with human IgG at the designed ELISA method. Rabbit IgG was purified by Ion-Exchange Chromatography (IEC) method. Purity was assessed by SDS-PAGE method. In non-reduced condition only one band was seen in about 150kDa MW position and in reduced form, two bands were seen in 50 and 25kDa MW positions. Rabbit IgG was digested by pepsin enzyme. The antibody fragments solution was applied to Gel filtration column to isolate the F(ab')2. Non-reduced SDS-PAGE for determining the purity of F(ab')2 fragment resulted in one band in 100kDa corresponds to F(ab')2 fragment and a band in 150kDa MW position corresponds to undigested IgG antibodies. The activities of FITC conjugated F(ab')2 fragment and commercial ones were compared using flowcytometry method. The activity results implied that the FITC conjugated- anti human F(ab')2 fragment worked as efficiently as the commercial one.

Correction: Cysteine modification reveals an allosteric inhibitory site on the CAL PDZ domain.

Bioscience Reports (2018)https://doi.org/10.1042/BSR20180231After acceptance and the publication of the Accepted 3, follow-up experiments revealed unexpected variability in the binding constants obtained by the mixed-KD analysis of FP binding data as shown in Table 2 in the Accepted Manuscript. Further investigation using non-reducing SDS–PAGE found previously undetected PDZ–PDZ dimerization triggered by extended incubation with the small-molecule compounds under stringent labeling conditions. This dimerization is not accounted for by our mixed-KD model. The final Version of Record instead reports apparent KD values determined from FP titrations with PDZ domains treated to minimize PDZ–PDZ dimerization while preserving high levels of small-molecule adduct formation, together with associated changes in text.This has been approved by the Editorial Board of Bioscience Reports.

Unfolding of IgG domains detected by non-reducing SDS-PAGE

Monoclonal antibodies are very important in modern therapeutics and constitute a substantial percentage of newly approved drugs. Every therapeutic monoclonal antibody must be analyzed for structural and functional integrity, and all protein heterogeneities need to be identified and quantified. The conformational stabilities of the monoclonal antibodies are also important for antibody storage and handling stabilities. One of the first and simplest of the structural analysis techniques utilized is SDS-PAGE, which can be performed both with and without prior reduction to break disulfide bonds. This permits sizing of both heavy and light chains in the reduced condition, and sizing of the intact antibody and any disulfide aggregates in the non-reduced condition. Analyzing our human anti-cocaine monoclonal antibody, we noted unexpectedly larger apparent molecular weights and apparent protein size heterogeneities using non-reducing SDS-PAGE. These apparent molecular weight heterogeneities are not consistent with other analysis techniques. Heterogeneities were noted using several heating and pre-electrophoretic sample preparation protocols, and are modified by the inclusion of small concentrations of certain alcohols such as propanol and butanol. All of these unexpected results were also observed for a commercial human IgG1 antibody, suggesting that these observations are applicable to IgGs in general. Thus, careful attention must be paid to the interpretation of non-reducing SDS-PAGE results for IgGs. It is hypothesized that differential thermal unfolding of the Fab, CH2 and CH3 domains of the IgGs in SDS give rise to the stable, discrete bands observed using different heating protocols prior to non-reducing SDS-PAGE.