Identification and Activity Study of an Impurity Band Observed in the nrSDS-PAGE of Aflibercept.

BACKGROUND: Aflibercept is a biopharmaceutical targeting vascular endothelial growth factor (VEGF) that has shown promise in the treatment of neovascular age-related macular degeneration (nAMD) and diabetic macular edema (DME) in adults. Quality control studies of aflibercept employing non-reduced SDS-PAGE (nrSDS-PAGE) have shown that a significant variant band (IM1) is consistently present below the main band. Considering the quality control strategy of biopharmaceuticals, structural elucidation and functional studies are required. METHODS: In this study, the variant bands in nrSDS-PAGE were collected through electroelution and identified by peptide mass fingerprinting based on liquid chromatography-tandem MS (LC-MS/MS). This variant was expressed using knob-into-hole (KIH) design transient transfection for the detection of ligand affinity, binding activity and biological activity. RESULTS: The variant band was formed by C-terminal truncation at position N99 of one chain in the aflibercept homodimer. Then, this variant was successfully expressed using KIH design transient transfection. The ligand affinity of the IM1 truncated variant was reduced by 18-fold, and neither binding activity nor biological activity were detected. CONCLUSIONS: The efficacy of aflibercept is influenced by the loss of biological activity of the variant. Therefore, this study supports the development of a quality control strategy for aflibercept.

Gel electrophoresis/electroelution sorting fractionator combined with filter-aided sample preparation for deep proteomic analysis.

Sample preparation and protein fractionation are important issues for proteomic studies. Protein extraction procedures strongly affect the performance of fractionation methods by provoking protein dispersion in several fractions. The most notable exception is the gel-based electrophoretic protein fractionation due to its resolution and effectiveness of sodium dodecyl sulfate as a solubilizing agent, while its main limitation lies in the poor recovery of the gel-trapped proteins. We created a fractionator device to separate complex mixture of proteins and peptides that is based on the continuous gel electrophoresis/electroelution sorting of these molecules. In an unsupervised process, complex mixtures of proteins or peptides are fractionated into the gel while separated fractions are simultaneously and sequentially electroeluted to the solution containing wells. The performance of the device was studied for protein fractionation in terms of reproducibility, protein recovery, and loading capacity. In a setup free of sodium dodecyl sulfate, complex peptide mixtures can also be fractionated. More than 11,700 proteins were identified in the whole-cell lysate of the CaSki cell line by using the fractionator combined with the filter-aided sample preparation method and mass spectrometry analysis. Fractionator-based proteome characterization increased 1.7-fold the number of identified proteins compared to the unfractionated sample analysis.

RNA electroelution: Comparing two electroeluter models.

RNA represents a vibrant area of research and many studies use techniques that require large amounts of purified RNA. One common purification method involves slicing a section of a polyacrylamide gel containing the RNA of interest and eluting the RNA out of the gel using electroelution. Various electroeluter models are available but sometimes a given model becomes discontinued, compelling researchers to choose a different model. Here, we have compared two electroeluters with different chamber designs for their ability to recover RNA from gel pieces. Our results show that both electroeluters are effective and recover comparable amounts of purified RNA.

An innovative ring-shaped electroeluter for high concentration preparative isolation of protein from polyacrylamide gel.

A ring-shaped electroeluter (RSE) was designed for protein recovery from polyacrylamide gel matrix. The RSE was designed in such a way that a ring-shaped well was used to place gel slices and an enrichment well was used to collect eluted protein samples. With HSA as model protein, the electroelution time was less than 30 min with 80% recovery rate, and the concentration of recovered protein was 50 times higher than that of conventional method. The RSE could be reused at least ten times. The developed device makes great advance towards economic electroelution of biomolecules (such as proteins) from gel matrix.

Gel Protein Extraction's Impact on Conformational Epitopes of Linear Non-Tagged MPT64 Protein.

The production and purification of recombinant proteins are crucial to acquiring pure MPT64 protein. Due to the fact that protein epitopes may undergo conformational changes during purification, this study, therefore, investigated an effective rapid purification method to produce highly intracellular pure MPT64 protein without causing conformational changes in the epitope under denaturing conditions. MPT64 was isolated from E. coli and electrophoresed using gel SDS-PAGE. Then, the desired protein bands were excised and purified with two methods: electroelution and passive elution. The isolated protein was identified via peptide mass fingerprinting using MALDI-TOF MS and reacted with IgG anti-MPT64, and the cross-reactivity of the isolated protein with IgY anti-MPT64 was confirmed using Western blot. The results show that both of these methods produced pure MPT64 protein, and the MPT64 protein was confirmed based on the MALDI-TOF MS results. Neither of these two methods resulted in epitope changes in the MPT64 protein so it could react specifically with both antibodies. The yield of MPT64 protein was higher with electroelution (2030 ± 41 µg/mL) than with passive elution (179.5 ± 7.5 µg/mL). Thus, it can be inferred that the electroelution method is a more effective method of purifying MPT64 protein and maintaining its epitope than the passive elution method.

Application of an electro elution system for direct purification of linear covalently closed DNA fragments.

Gene therapy is a powerful treatment modality. Non-viral gene therapy vectors power one arm of this important approach, due to their enhanced safety profile compared to their viral counterparts. New non-viral approaches continue to be developed, but purification can bottleneck the scaleup and cost-effectiveness and quality of some of these advanced vectors. We require more advanced purification and separation techniques compared to conventional methods to maximize resolution in a scalable manner. The Prep Cell system is a continuous electro elution system that contains a circular gel casting tube where DNA mixtures can be run through and subsequently migrate into an elution chamber, to be eluted by a peristaltic pump. This DNA separation and purification process confers advantages over other conventional methods, including i) the elimination of multiple downstream purification process requirements; ii) its ability to be applied in mid-scale settings, and iii), its high-resolution power. In this study, we assessed the ability of this Prep Cell Model 491 system to purify a novel type of non-viral linear covalently closed (LCC) DNA minivector (ministring DNA) from its precursor parent plasmid DNA and process by-product DNA species by analyzing for effective separation via agarose gel electrophoresis, recovery yield, single enzyme digestion, and quality control assessments. Overall, effective separation and resolution of mini-DNA vectors was obtained using the Prep Cell system, conferring its potential to be applied towards mid-scale purification of DNA vectors for a variety of research, and eventually, clinical applications.

Preparation of Chemically Modified DNA Library for SELEX via Incorporation of CLB-dUTP in Primer Extension Reaction.

Aptamers are single-stranded DNA or RNA molecules which bind with high specificity to the molecular target for which they have been selected. Through a number of unnatural modifications, the diversity of interactions available for this class of molecules can be greatly expanded, potentially leading to better binding properties. Herein we describe a method to prepare an initial chemically modified DNA library for SELEX. It comprises the synthesis of a modified nucleotide in the CuAAC reaction and its purification by an adapted HPLC method. Subsequent stage is the incorporation of the prepared modified nucleotide into a DNA library in a primer extension reaction and a quick and easy method of its purification using an electroelution device. This allows the recovery of the resulting chemically modified ssDNA library with high efficiency to kick off the aptamer selection process.

Production, purification and physicochemical characterization of D-xylose/glucose isomerase from Escherichia coli strain BL21.

Cell lysate of Escherichia coli strain BL21 showed significant D-glucose isomerase activity. The rate of glucose conversion was increased up to 40% when cells were induced with 1% D-xylose. E. coli BL21 xylose isomerase (ECXI-BL21) was purified to homogeneity, up to 1.9-fold with overall 10.88% enzyme yield by heat shock, salting out and electro-elution. The molecular mass of ECXI-BL21 was estimated as 43.9 kDa on SDS-PAGE. pHopt. and Topt. of the enzyme were calculated as 7.0 and 50 °C, respectively. Activation energy (E a) of ECXI-BL21 was 45 kJ/mol. Enzyme was stable from 30 to 55 °C and at pH range 6.0-8.0. ECXI-BL21(holo) was activated by 10 mM magnesium (35%), 0.5 mM cobalt (20%) and manganese (25%), and 0.5/10 mM Mn2+/Mg2+ (50%) and Co2+/Mg2+ (30%) as compared to ECXI-BL21(apo). Catalytic affinity (K m) of ECXI-BL21 for D-glucose was calculated as 0.82 mM, while maximum velocity (V max) of the reaction D-glucose(aldo) ⇌ D-fructose(keto) was 108 µmol/mg/min. D-fructose formed was identified on silica gel plate. This thermophilic enzyme, T m = 75 °C, has great potential for high fructose syrup production used in food and soft drink industries.

Protein Extraction from Gels: A Brief Review.

Protein gel electrophoresis is an important procedure carried out in protein studies. Elution and recovery of proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) are often necessary for further downstream analyses. The process involves localizing the protein of interest on the gel following SDS-PAGE, eluting the protein from the gel, removing SDS from the eluted sample, and finally renaturing the protein (e.g., enzymes) for subsequent analyses. Investigators have extracted proteins from gels by a variety of techniques. These include dissolution of the gel matrix, passive diffusion, and electrophoretic elution. Proteins eluted from gels have been used successfully in a variety of downstream applications, including protein chemistry, proteolytic cleavage, determination of amino acid composition, polypeptide identification by trypsin digestion and matrix-assisted laser desorption ionization-time of flight mass spectroscopy, as antigens for antibody production, identifying a polypeptide corresponding to an enzyme activity, and other purposes. Protein yields ranging from nanogram levels to 100 µg have been obtained. Here, we review some of the methods that have been used to elute proteins from gels.

A Detailed Protein-SELEX Protocol Allowing Visual Assessments of Individual Steps for a High Success Rate.

As a nucleic acid alternative to traditional antibody, aptamer holds great potential in various fields of biology and medicine such as targeted gene therapy, drug delivery, bio-sensing, and laboratory medicine. Over the past decades, the conventional Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method has undergone dramatic modifications and improvements owing to developments in material sciences and analytical techniques. However, many of the recently developed strategies either require complex materials and instruments or suffer from low efficiency and high failure rates in the selection of desired aptamers. Accordingly, the development of aptamers against new or novel targets is still a major obstacle for aptamer-based research and application. Here, an improved protein-SELEX procedure is presented for simplified and highly efficient isolation of aptamers against protein targets. Approaches are described that ensure a high success rate in aptamer selection by simplifying polymerase chain reaction procedures, introducing denature gel, utilizing an electro-elution-based single-stranded DNA separation strategy, as well as an enzyme-linked immunosorbent assay-based highly sensitive binding assay. In addition, a simplified sample preparation method for MiSeq-based next-generation sequencing is also introduced. While a recombinant protein as a bait protein for SELEX is discussed here, this protocol will also be invaluable for researchers wishing to develop aptamers against targets other than proteins such as small molecules, lipids, carbohydrates, cells, and micro-organisms for future gene therapy and/or diagnostics.

Revisit of Imidazole-Zinc Reverse Stain for Protein Polyacrylamide Gel Electrophoresis.

Imidazole-zinc reverse stain (ZN stain) is known for high sensitivity, ease of use, and cost-effective feature. ZN stain is compatible to many experiments of which those are proteomics-related in particular. Here, we describe the ZN staining procedures and the subsequent procedures incorporated in detail, along with the improvements of setup in aspects of visualization and documentation for postprocessing ZN stained gel images.

Enzymatic Synthesis and Fractionation of Fluorescent PolyU RNAs.

The physical properties of viral-length polyuridine (PolyU) RNAs, which cannot base-pair and form secondary structures, are compared with those of normal-composition RNAs, composed of comparable numbers of each of A, U, G and C nucleobases. In this protocol, we describe how to synthesize fluorescent polyU RNAs using the enzyme polynucleotide phosphorylase (PNPase) from Uridine diphosphate (UDP) monomers and how to fractionate the polydisperse synthesis mixture using gel electrophoresis, and, after electroelution, how to quantify the amount of polyU recovered with UV-Vis spectrophotometry. Dynamic light scattering was used to determine the hydrodynamic radii of normal-composition RNAs as compared to polyU. It showed that long polyU RNAs behave like linear polymers for which the radii scale with chain length as N1/2, as opposed to normal-composition RNAs that act as compact, branched RNAs for which the radii scale as N1/3.

Antibacterial Activity of Isolated Immunodominant Proteins of Naja Naja (Oxiana) Venom.

The aim of this study is to investigate antibacterial effects of immunodominant proteins isolated from the venom of Naja Naja Oxiana snake against Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa. The innate immune system is an important line of defense against bacterial diseases. Antibacterial peptides and proteins produced by snake venoms have recently attracted significant attention due to their relevance to bacterial diseases and the potential of being converted into new therapeutic agents. Identification of immunodominant proteins of the venom of Naja Naja Oxiana snake was performed by SDS-PAGE and western blot analysis. Identified proteins were isolated directly from preparative gel electrophoresis by Electro-elution. In the next step, antibacterial effects of immunodominant proteins were tested against several strains of clinical isolates, including S.aureus, B.subtilis (Gram-positive bacteria) P.aeruginosa and E.coli (Gram-negative bacteria) using broth microdilution and disc-diffusion assays. In order to compare the results of the disc-diffusion assay, antibacterial effects of several antibiotics (Gentamicin, Ampicillin, Penicillin, Amoxicillin and Ciprofloxacin) were also examined using the same conditions. Results showed that immunodominant proteins of (14, and 65kDa) with high immunogenicity were very effective in inhibiting the growth of two Gram-positive bacteria (S.aureus, B.sub) that were tested. However, they were only moderately effective in inhibiting the growth of the two tested Gram-negative bacteria (P.aeruginosa and E.coli). However, immunodominant proteins of 22 kDa and 32kDa with high immunogenicity, showed slight effectiveness in inhibiting the growth of two; the Gram-positive and Gram-negative bacteria that were tested. To the best of our knowledge, these immunodominant proteins are novel antigens for potent antimicrobial effects against two gram-positive bacteria (S.aureus, B.subtilis ) and less antimicrobial effect against two gram-negative bacteria (E.coli, P.aeruginosa) that were prepared .

A simple electroelution method for rapid protein purification: isolation and antibody production of alpha toxin from Clostridium septicum.

Clostridium septicum produces a number of diseases in human and farm animals which, in most of the cases, are fatal without clinical intervention. Alpha toxin is an important agent and the unique lethal virulent factor produced by Clostridium septicum. This toxin is haemolytic, highly lethal and necrotizing activities but is being used as an antigen to develop animal vaccines. The aim of this study was to isolate the alpha toxin of Clostridium septicum and produce highly specific antibodies against it. In this work, we have developed a simple and efficient method for alpha toxin purification, based on electroelution that can be used as a time-saving method for purifying proteins. This technique avoids contamination by other proteins that could appear during other protein purification techniques such chromatography. The highly purified toxin was used to produce polyclonal antibodies. The specificity of the antibodies was tested by western blot and these antibodies can be applied to the quantitative determination of alpha toxin by slot blot.

Experimental setup for the identification of mitochondrial protease substrates by shotgun and top-down proteomics.

Mitochondria possess a proteolytic system that contributes to the regulation of mitochondrial dynamics, mitochondrial biogenesis and mitophagy. We aimed at the identification by bottom-up proteomics of altered protein processing due to the activation of mitochondrial proteases in a cellular model of impaired dopamine homeostasis. Moreover, we optimized the conditions for top-down proteomics to identify the cleavage site sequences.

RNA purification by preparative polyacrylamide gel electrophoresis.

Preparative polyacrylamide gel electrophoresis (PAGE) is a powerful tool for purifying RNA samples. Denaturing PAGE allows separation of nucleic acids that differ by a single nucleotide in length. It is commonly used to separate and purify RNA species after in vitro transcription, to purify naturally occurring RNA variants such as tRNAs, to remove degradation products, and to purify labeled RNA species. To preserve RNA integrity following purification, RNA is usually visualized by UV shadowing or stained with ethidium bromide or SYBR green dyes.

Preparative SDS-PAGE Electroelution for Rapid Purification of Alkyl Hydroperoxide Reductase from Helicobacter pylori.

BACKGROUND: Alkyl hydroperoxide reductase (AhpC) of Helicobacter pylori is considered as a diagnostic antigen. Therefore, this antigen can be used to detect H. pylori infection by stool immunoassays such as ELISA. The aim of this study was to simplify the AhpC protein purification procedures. METHODS: For whole cell protein extraction, the bacterial cells were ruptured by octly-ß-D glucopyranoside. The isolation and purification of AhpC protein were attempted by various techniques including ammonium sulfate precipitation, dialysis, preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroelution. RESULTS: A simple method was used for protein purification AhpC protein. One-dimensional preparative gel electrophoresis allows a single and short purification step; the high resolution capacity of this technique leads to a high level of purity of the protein. Moreover, it avoids contamination by other non-specific proteins which often appear during protein purification by column chromatography. CONCLUSION: The present method is simple, rapid and makes it possible to preparate AhpC from H. pylori.