Single-step Purification Research Articles

One-Step Purification of Melittin Derived from Apis mellifera Bee Venom.

The concern over the use of melittin in honey bee venom due to its adverse reaction caused by allergens such as phospholipase A2 (PLA2) and hyaluronidase (HYA) has been an obstacle towards its usage. We developed a novel single-step method for melittin purification and the removal of PLA2 and HYA. This study explores the influence of pH, buffer compositions, salt concentration, and types of cation-exchange chromatography resins on the recovery of melittin and the removal of both HYA and PLA2. Melittin was readily purified with a strong cation-exchange resin at pH 6.0 with sodium phosphate buffer. It resulted in a recovery yield of melittin up to 93% (5.87 mg from a total of 6.32 mg of initial melittin in crude bee venom), which is higher than any previously reported studies on melittin purification. PLA2 (99%) and HYA (96%) were also successfully removed. Our study generates a single-step purification method for melittin with a high removal rate of PLA2 and HYA, enabling melittin to be fully utilized for its therapeutic purposes.

Single-Step Affinity Purification of ERK Signaling Complexes Using the Streptavidin-Binding Peptide (SBP) Tag.

Elucidation of biological functions of signaling proteins is facilitated by studying their protein-protein interaction networks. Affinity purification combined with mass spectrometry (AP-MS) has become a favorite method to study protein complexes. Here we describe a procedure for single-step purification of ERK (Rolled) and associated proteins from Drosophila cultured cells. The use of the streptavidin-binding peptide (SBP) tag allows for a highly efficient isolation of native ERK signaling complexes, which are suitable for subsequent analysis by mass spectrometry. Our analysis of the ERK interactome has identified both known and novel signaling components. This method can be easily adapted for SBP-based purification of protein complexes in any expression system.

Integrated process for the purification and immobilization of the envelope protein domain III of dengue virus type 2 expressed in Rachiplusia nu larvae and its potential application in a diagnostic assay

Dengue incidence has grown dramatically in the last years, with about 40% of the world population at risk of infection. Recently, a vaccine developed by Sanofi Pasteur has been registered, but only in a few countries. Moreover, specific antiviral drugs are not available. Thus, an efficient and accurate diagnosis is important for disease management. To develop a low-cost immunoassay for dengue diagnosis, in the present study we expressed the envelope protein domain III of dengue virus type 2 in Rachiplusia nu larvae by infection with a recombinant baculovirus. The antigen was expressed as a fusion to hydrophobin I (DomIIIHFBI) to easily purify it by an aqueous two-phase system (ATPS) and to efficiently immobilize it in immunoassay plates. A high level of recombinant DomIIIHFBI was obtained in R. nu, where yields reached 4.5 mg per g of larva. Also, we were able to purify DomIIIHFBI by an ATPS with 2% of Triton X-114, reaching a yield of 73% and purity higher than 80% in a single purification step. The recombinant DomIIIHFBI was efficiently immobilized in hydrophobic surface plates. The immunoassay we developed with the immobilized antigen was able to detect IgG specific for dengue virus type 2 in serum samples and not for other serotypes.

Enhancement of solubility, purification and inclusion-bodies-refolding of an active pectin lyase from Penicillium occitanis expressed in Escherichia coli

Pectin lyase (pnl) is the only pectinase able to hydrolyze directly the highly methylated pectin without liberating the toxic methanol and without disturbing ester content responsible for specific aroma of juices. The cDNA of Penicillium occitanis pnl (mature form) was cloned into pET-21a as expression vector and over-expressed into Esherichia coli. Most of recombinant pnl was expressed as inclusion bodies. Pnl activity was confirmed by colorimetric assay. To enhance the solubility yield of the expressed pnl, the effects of induction temperature, host strain and expression level were optimized. Maximal production of functional pnl was obtained after induction by 0.4mM IPTG at 30°C and 150rpm for 16h. Interestingly, the use of Origami host strain, having an oxidized cytoplasm favoring disulfide bonds formation required for the active conformation of the enzyme, has significantly improved the yield of the soluble active form of recombinant pnl. This pnl was successfully purified through a single step purification using His-Trap affinity column chromatography. This work is the first to report pnl expression into Origami strain. Alternatively, the inclusion bodies were isolated, denatured by high concentration of urea and gradually refolded by successive dialysis, leading to their transformation into soluble and active form.

A Penicillium chrysogenum-based expression system for the production of small, cysteine-rich antifungal proteins for structural and functional analyses.

BackgroundSmall, cysteine-rich and cationic antifungal proteins (APs) from filamentous ascomycetes, such as NFAP from Neosartorya fischeri and PAF from Penicillium chrysogenum, are promising candidates for novel drug development. A prerequisite for their application is a detailed knowledge about their structure–function relation and mode of action, which would allow protein modelling to enhance their toxicity and specificity. Technologies for structure analyses, such as electronic circular dichroism (ECD) or NMR spectroscopy, require highly purified samples and in case of NMR milligrams of uniformly 15N-/13C-isotope labelled protein. To meet these requirements, we developed a P. chrysogenum-based expression system that ensures sufficient amount and optimal purity of APs for structural and functional analyses.ResultsThe APs PAF, PAF mutants and NFAP were expressed in a P. chrysogenum ∆paf mutant strain that served as perfect microbial expression factory. This strain lacks the paf-gene coding for the endogenous antifungal PAF and is resistant towards several APs from other ascomycetes. The expression of the recombinant proteins was under the regulation of the strong paf promoter, and the presence of a paf-specific pre-pro sequence warranted the secretion of processed proteins into the supernatant. The use of defined minimal medium allowed a single-step purification of the recombinant proteins. The expression system could be extended to express PAF in the related fungus Penicillium digitatum, which does not produce detectable amounts of APs, demonstrating the versatility of the approach. The molecular masses, folded structures and antifungal activity of the recombinant proteins were analysed by ESI–MS, ECD and NMR spectroscopy and growth inhibition assays.ConclusionThis study demonstrates the implementation of a paf promoter driven expression cassettes for the production of cysteine-rich, cationic, APs in different Penicillium species. The system is a perfect tool for the generation of correctly folded proteins with high quality for structure–function analyses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0586-4) contains supplementary material, which is available to authorized users.

Expression and characterization of thermotolerant lipase with broad pH profiles isolated from an Antarctic Pseudomonas sp strain AMS3.

A gene encoding a thermotolerant lipase with broad pH was isolated from an Antarctic Pseudomonas strain AMS3. The recombinant lipase AMS3 was purified by single-step purification using affinity chromatography, yielding a purification fold of approximately 1.52 and a recovery of 50%. The molecular weight was approximately ∼60 kDa including the strep and affinity tags. Interestingly, the purified Antarctic AMS3 lipase exhibited broad temperature profile from 10–70 °C and stable over a broad pH range from 5.0 to pH 10.0. Various mono and divalent metal ions increased the activity of the AMS3 lipase, but Ni2+ decreased its activity. The purified lipase exhibited the highest activity in the presence of sunflower oil. In addition, the enzyme activity in 25% v/v solvents at 50 °C particularly to n-hexane, DMSO and methanol could be useful for catalysis reaction in organic solvent and at broad temperature.

Single-step purification and characterization of an extreme halophilic, ethanol tolerant and acidophilic xylanase from Aureobasidium pullulans NRRL Y-2311-1 with application potential in the food industry.

An extracellular xylanase from Aureobasidium pullulans NRRL Y-2311-1 produced on wheat bran was purified by a single-step chromatographic procedure. The enzyme had a molecular weight of 21.6kDa. The optimum pH and temperature for xylanase activity were 4.0 and 30-50°C, respectively. The enzyme was stable in the pH range of 3.0-8.0. The inactivation energy of the enzyme was calculated as 218kJmol-1. The xylanase was ethanol tolerant and kept complete activity in the presence of 10% ethanol. Likewise, it retained almost complete activity at a concentration range of 0-20% NaCl. In general, the enzyme was resistant to several metal ions and reagents. Mg2+, Zn2+, Cu2+, K1+, EDTA and β-mercaptoethanol resulted in enhanced xylanase activity. The Km and Vmax values on beechwood xylan were determined to be 19.43mgml-1 and 848.4Uml-1, respectively. The enzyme exhibits excellent characteristics and could, therefore, be a promising candidate for application in food and bio-industries.

Gene cloning, expression and polyclonal antibody preparation of Rab3A for protein interaction analysis.

BackgroundRab3A is a GTP-binding protein and plays critical roles in the regulation of synaptic vesicle exocytosis. Up to date, how Rab3A participates in such a regulatory process is not completely clear.ResultsIn this report the Rab3A gene from Rattus norvegicus was cloned and heterologously expressed in E. coli using pCold-TF expression vector with folding capacity. Due to the presence of His-tag sequence on the N-terminal side, Rab3A fusion protein was purified to greater than 95 % purity with a single Ni-affinity purification step. After the Rab3A fusion protein was used to immunize mice, an anti-serum against Rab3A with a titer of about 6000 was generated. Western blot analysis indicated that the prepared polyclonal antibody could recognize both Rab3A fusion protein and native Rab3A protein. To remove the tag sequence, thrombin was used to cleave the Rab3A fusion protein, followed by SDS-PAGE to separate the cleavage products. Using the gel protein recovery strategy with a Micro Protein PAGE Recovery Kit, the de-tagged Rab3A protein of electrophoretic purity was prepared.ConclusionsThe present work not only prepared the ground for the study on Rab3A-mediated protein interactions, but also provided systematic experimental methods referable for the similar studies.

Characterization of dietary fucoxanthin from Himanthalia elongata brown seaweed

This study explored Himanthalia elongata brown seaweed as a potential source of dietary fucoxanthin which is a promising medicinal and nutritional ingredient. The seaweed was extracted with low polarity solvents (n-hexane, diethyl ether, and chloroform) and the crude extract was purified with preparative thin layer chromatography (P-TLC). Identification, quantification and structure elucidation of purified compounds was performed by LC-DAD-ESI-MS and NMR (1H and 13C). P-TLC led purification yielded 18.6mg/g fucoxanthin with 97% of purity based on the calibration curve, in single-step purification. LC-ESI-MS (parent ion at m/z 641 [M+H-H2O]+) and NMR spectra confirmed that the purified band contained all-trans-fucoxanthin as the major compound. Purified fucoxanthin exhibited statistically similar (p>0.05) DPPH scavenging capacity (EC50: 12.9μg/mL) while the FRAP value (15.2μg trolox equivalent) was recorded lower (p<0.05) than the commercial fucoxanthin. The promising results of fucoxanthin purity, recovery and activity suggested that H. elongata seaweed has potential to be exploited as an alternate source for commercial fucoxanthin production.

High-resolution, preparative purification of PEGylated protein using a laterally-fed membrane chromatography device.

We discuss the use of a laterally-fed membrane chromatography (or LFMC) device for single-step purification of mono-PEGylated lysozyme. Recent studies have shown such LFMC devices to be suitable for high-resolution, multi-component separation of proteins in the bind-and-elute mode. The device used in this study contained a stack of rectangular cation-exchange membranes having 9.25mL bed volume. PEGylation of lysozyme was carried out in batch mode using 5kDa methoxy-polyethyleneglycol propionaldehyde (or m-PEG propionaldehyde) in the presence of sodium cyanoborohydride as reducing agent. Membrane chromatographic separation was carried out at 1.62 membrane bed volumes per minute flow rate, in the bind-and-elute mode. When a salt gradient was applied, the higher PEGylated forms of lysozyme (i.e. the byproducts) eluted earlier than mono-PEGylated lysozyme (the target product), while lysozyme eluted last. Under elution conditions optimized for resolution and speed, the separation could be carried out in less than 15 membrane bed volumes. High purity and recovery of mono-PEGylated lysozyme was obtained. The resolution of separation of mono-PEGylated lysozyme obtained under the above condition was comparable to that reported in the literature for equivalent cation-exchange resin columns while the flow rate expressed in bed volumes/min was 21.7 times higher. Also, the number of theoretical plates per meter was significantly higher with the LFMC device. Therefore the LFMC based purification process discussed in this paper combined high-productivity with high-resolution.

Recombinant overexpression of camel hepcidin cDNA in Pichia pastoris: purification and characterization of the polyHis‐tagged peptide HepcD‐His

Hepcidin, a liver-expressed antimicrobial peptide, has been demonstrated to act as an iron regulatory hormone as well as to exert a wide spectrum of antimicrobial activity. The aim of this work was the expression, as secreted peptide, purification, and characterization of a new recombinant polyHis-tagged camel hepcidin (HepcD-His) in yeast Pichia pastoris. The use of this eukaryotic expression system, for the production of HepcD-His, having 6 histidine residues at its C terminus, was simpler and more efficient compared with the use of the prokaryotic system Escherichiacoli. Indeed, a single purification step was required to isolate the soluble hepcidin with purity estimated more that 94% and a yield of 2.8 against 0.2mg/L for the Ecoli system. Matrix-assisted laser desorption/ionization time-of-flight (TOF)/TOF mass spectrometry of the purified HepcD-His showed 2 major peaks at m/z 4524.64 and 4634.56 corresponding to camel hepcidin with 39 and 40 amino acids. Evaluation of disulfide bond connectivity with the Ellman method showed an absence of free thiol groups, testifying that the 8 cysteine residues in the peptide are displayed, forming 4 disulfide bridges. Circular dichroism spectroscopy showed that camel hepcidin structure was significantly modified at high temperature of 90°C and returns to its original structure when incubation temperature drops back to 20°C. Interestingly, this peptide showed also a greater bactericidal activity, at low concentration of 9.5μM, against Ecoli, than the synthetic analog DH3. Thus, the production, at a large scale, of the recombinant camel hepcidin, HepcD-His, may be helpful for future therapeutic applications including bacterial infection diseases.

Simplified Method to Produce Human Bioactive Leukemia Inhibitory Factor in Escherichia coli

Background: Human leukemia inhibitory factor (hLIF) is a poly functional cytokine with numerous regulatory effects on different cells. Main application of hLIF is maintaining pluripotency of embryonic stem cells. hLIF indicated effective work in implantation rate of fertilized eggs and multiple sclerosis (MS) treatment. Low production of hLIF in eukaryotic cells and prokaryotic host’s problems for human protein production convinced us to develop a simple way to reach high amount of this widely used clinical and research factor. Objectives: In this study we want to purify recombinant human leukemia inhibitory factor in single simple method. Materials and Methods: This is an experimental study, gene expression: human LIF gene was codon optimized for expression in Escherichia coli and attached his-tag tail to make it extractable. After construction and transformation of vector to E. coli, isopropyl β-D-1-thiogalactopyranoside (IPTG) used for induction. Single step immobilized metal affinity chromatography (IMAC) used for purification confirmed by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and western blotting. Bioactivity of the hLIF were tested by MTT assay with TF-1 cells and CISH gene stimulation in monocyte and TF-1 by real-time PCR. Induction by 0.4 mM of IPTG in 25°C for 3 hours indicated best result for soluble expression. SPSS indicated P ˂ 0.05 that is significant for our work. Results: Cloning, expression, and extraction of bio active rhLIF was successfully achieved according MTT assay and real time PCR after treatment of TF-1 and monocyte cell lines. Conclusions: We developed an effective single step purification method to produce bioactive recombinant hLIF in E. coli. For the first time we used CISH gene stimulating for bioactivity test for qualifying of recombinant hLIF for application.

Mineralogical Analysis and Mechano-Chemical Purification of Natural Silica Ore for High Purity Silica Powder

To produce 4N grade high-purity silica powder from natural ore, the mineralogical characteristics of natural silica ore were investigated and their effects on the purification process were revealed. The Chinese silica mineral ore used was composed of iron and aluminum as main impurities and calcium, magnesium, potassium, sodium, and titanium as trace impurities; these trace impurities generally exist as either single oxides or complex oxides. It was confirmed that liberation and acidic washing of the impurities were highly dependent on the particle size of the ground silica ore and on its mineralogical characteristics such as the distribution and phases of existing impurities. It is suggested that appropriate size reduction of silica ore should be realized for optimized purification according to the origin of the natural silica ore. A single step purification process, the mechano-chemical washing (MCW) process, was proposed and verified in comparison with the conventional multi step washing process.

Single step purification of asparaginase from endophytic bacteria Pseudomonas oryzihabitans exhibiting high potential to reduce acrylamide in processed potato chips

This study reports the purification and characterization of extracellular asparaginase from an endophytic bacteria Pseudomonas oryzihabitans isolated from Hibiscus rosa-sinensis. Sixty endophytes isolated from medicinal plants H. rosa-sinensis, Withania somnifera and Ocimum sanctum were screened for their potential to produce asparaginase. Twenty seven isolates were found positive and exhibited asparaginase activity in the range of 0.6–2.3U/mL. Isolate Hib-3 produced maximal level of asparaginase (2.1U/mL) which was identified as P. oryzihabitans. The isolate under optimized culture conditions produced 4.3U/mL of asparaginase with productivity of 238.9U/L/h. Single step purification of asparaginase was achieved using SP sephadex cation exchange column. The purified asparaginase showed specific activity of 39μmolmin−1mgprotein−1 corresponding to 42 fold purification. Electrophoretic analysis indicated asparaginase to be heterodimer showing two bands of 54 and 50kDa on SDS-PAGE, whereas, single band of 208.7kDa was observed on Native-PAGE. Purified asparaginase was optimally active at pH 8.0 and 37–40°C. Addition of purified asparaginase (2.8U/g dried potato), reduced acrylamide (90%) in fried potatoes. In addition, the asparaginase was also found to exhibit anti-proliferative activity against MCF7-Breast and HeLa-Cervix carcinoma cell lines. This is the first report on purification of asparaginase from endophytic bacteria P. oryzihabitans isolated from H. rosa-sinensis.

A low-temperature polygalacturonase from P. occitanis: characterization and application in juice clarification

An extracellular endo-polygalacturonase (PGase) was purified, after a single purification step, from the constitutive and hyperpectinolytic CT1 mutant of Penicillium occitanis. This enzyme named PG2 has a molecular weight of 42kDa. It was optimally active at 35°C and pH6 with more than 85% of activity at pH7 in contrast to the majority of fungal PGase, generally acting at 50°C and pH5. The specific activity obtained was among the highest ones, 31397.26U/mg. The PGase activity increased with the decrease of the degree of methylation (DM) of pectin, but it was also able to degrade the highly methyl-esterified substrates, 70% (DM) and 90% (DM), with almost 80% and 40% of residual activity respectively. Interestingly, PG2 is completely inhibited by DEPC, suggesting the implication of a Histidine residue in the active site. The sequencing of P. occitanis whole genome allowed us to identify the pga2 gene encoding PG2 and to localize the His residue, target of DEPC, while it was absent in the PG1 that resisted to DEPC. Besides that, the potentialities of PG2 have been put in use in juice clarification of pear, banana and citrus juice.

Preparation of ribosomes for smFRET studies: A simplified approach

During the past decade, single-molecule studies of the ribosome have significantly advanced our understanding of protein synthesis. The broadest application of these methods has been towards the investigation of ribosome conformational dynamics using single-molecule Förster resonance energy transfer (smFRET). The recent advances in fluorescently labeled ribosomes and translation components have resulted in success of smFRET experiments. Various methods have been employed to target fluorescent dyes to specific locations within the ribosome. Primarily, these methods have involved additional steps including subunit dissociation and/or full reconstitution, which could result in ribosomes of reduced activity and translation efficiency. In addition, substantial time and effort are required to produce limited quantities of material. To enable rapid and large-scale production of highly active, fluorescently labeled ribosomes, we have developed a procedure that combines partial reconstitution with His-tag purification. This allows for a homogeneous single-step purification of mutant ribosomes and subsequent integration of labeled proteins. Ribosomes produced with this method are shown to be as active as ribosomes purified using classical methods. While we have focused on two labeling sites in this report, the method is generalizable and can in principle be extended to any non-essential ribosomal protein.

Modular Combination of Enzymatic Halogenation of Tryptophan with Suzuki–Miyaura Cross‐Coupling Reactions

AbstractThe combination of the biocatalytic halogenation of l‐tryptophan with subsequent chemocatalytic Suzuki–Miyaura cross‐coupling reactions leads to the modular synthesis of an array of C5, C6, or C7 aryl‐substituted tryptophan derivatives. In a three‐step one‐pot reaction, the bromo substituent is initially incorporated regioselectively by immobilized tryptophan 5‐, 6‐, or 7‐halogenases, respectively, with concomitant cofactor regeneration. The halogenation proceeds in aqueous media at room temperature in the presence of NaBr and O2. After the separation of the biocatalyst by filtration, a Pd catalyst, base, and boronic acid are added to the aryl halide formed in situ to effect direct Suzuki–Miyaura cross‐coupling reactions followed by tert‐butoxycarbonyl (Boc) protection. After a single purification step, different Boc‐protected aryl tryptophan derivatives are obtained that can, for example, be used for peptide or peptidomimetic synthesis.

PROLink-Single Step Circularization and Purification Procedure for the Generation of an Improved Variant of Human Growth Hormone.

Human growth hormone (hGH) plays an important role during human development and is also an approved therapeutic for the treatment of several diseases. However, one major drawback of hGH is its short circulating half-life requiring frequent administration, which is inconvenient and painful for the patients. Recent publications indicate that circularization greatly increases the stability of proteins due to their protection from exoproteolytic attack and a higher thermal stability of the circular form. Using sortase A, a transpeptidase isolated from Staphylococcus aureus, we developed a single step solid-phase circularization and purification procedure resulting in a circular version of hGH with improved properties. We could show that circular hGH binds to the recombinant hGH receptor with binding kinetics similar to those of linear hGH and that circularization does not alter the biological activity of hGH in vitro. Besides, circular hGH showed almost complete resistance toward exoproteolytic attack and slightly increased thermal stability which could possibly translate into an extended plasma half-life. The single step solid-phase circularization and purification procedure is in principle a generic process, which could also be applied for other proteins that meet the requirements for circularization.

ExtraPEG: A Polyethylene Glycol-Based Method for Enrichment of Extracellular Vesicles.

Initially thought to be a means for cells to eliminate waste, secreted extracellular vesicles, known as exosomes, are now understood to mediate numerous healthy and pathological processes. Though abundant in biological fluids, purifying exosomes has been challenging because their biophysical properties overlap with other secreted cell products. Easy-to-use commercial kits for harvesting exosomes are now widely used, but the relative low-purity and high-cost of the preparations restricts their utility. Here we describe a method for purifying exosomes and other extracellular vesicles by adapting methods for isolating viruses using polyethylene glycol. This technique, called ExtraPEG, enriches exosomes from large volumes of media rapidly and inexpensively using low-speed centrifugation, followed by a single small-volume ultracentrifugation purification step. Total protein and RNA harvested from vesicles is sufficient in quantity and quality for proteomics and sequencing analyses, demonstrating the utility of this method for biomarker discovery and diagnostics. Additionally, confocal microscopy studies suggest that the biological activity of vesicles is not impaired. The ExtraPEG method can be easily adapted to enrich for different vesicle populations, or as an efficient precursor to subsequent purification techniques, providing a means to harvest exosomes from many different biological fluids and for a wide variety of purposes.

Single-column purification of the tag-free, recombinant form of the neuronal calcium sensor protein, hippocalcin expressed in Escherichia coli

Hippocalcin is a 193 aa protein that is a member of the neuronal calcium sensor protein family, whose functions are regulated by calcium. Mice that lack the function of this protein are compromised in the long term potentiation aspect of memory generation. Recently, mutations in the gene have been linked with dystonia in human. The protein has no intrinsic enzyme activity but is known to bind to variety of target proteins. Very little information is available on how the protein executes its critical role in signaling pathways, except that it is regulated by binding of calcium. Further delineation of its function requires large amounts of pure protein. In this report, we present a single-step purification procedure that yields high quantities of the bacterially expressed, recombinant protein. The procedure may be adapted to purify the protein from inclusion bodies or cytosol in its myristoylated or non-myristoylated forms. MALDI-MS (in source decay) analyses demonstrates that the myristoylation occurs at the glycine residue. The protein is also biologically active as measured through tryptophan fluorescence, mobility shift and guanylate cyclase activity assays. Thus, further analyses of hippocalcin, both structural and functional, need no longer be limited by protein availability.