Ultrafast in-gel detection by fluorescent super-chelator probes with HisQuick-PAGE.

Polyacrylamide gel electrophoresis (PAGE) and immunoblotting (Western blotting) are the most common methods in life science. In conjunction with these methods, the polyhistidine-tag has proven to be a superb fusion tag for protein purification as well as specific protein detection by immunoblotting, which led to a vast amount of commercially available antibodies. Nevertheless, antibody batch-to-batch variations and nonspecific binding complicate the laborious procedure. The interaction principle applied for His-tagged protein purification by metal-affinity chromatography using N-nitrilotriacetic acid (NTA) was employed to develop small high-affinity lock-and-key molecules coupled to a fluorophore. These multivalent NTA probes allow specific detection of His-tagged proteins by fluorescence. Here, we report on HisQuick-PAGE as a fast and versatile immunoblot alternative, using such high-affinity fluorescent super-chelator probes. The procedure allows direct, fast, and ultra-sensitive in-gel detection and analysis of soluble proteins as well as intact membrane protein complexes and macromolecular ribonucleoprotein particles.

Recombinant production, purification, crystallization, and structure analysis of human transforming growth factor ß2 in a new conformation.

Transforming growth factor ß is a disulfide-linked dimeric cytokine that occurs in three highly related isoforms (TGFß1-TGFß3) engaged in signaling functions through binding of cognate TGFß receptors. To regulate this pathway, the cytokines are biosynthesized as inactive pro-TGFßs with an N-terminal latency-associated protein preceding the mature moieties. Due to their pleiotropic implications in physiology and pathology, TGFßs are privileged objects of in vitro studies. However, such studies have long been limited by the lack of efficient human recombinant expression systems of native, glycosylated, and homogenous proteins. Here, we developed pro-TGFß2 production systems based on human Expi293F cells, which yielded >2 mg of pure histidine- or Strep-tagged protein per liter of cell culture. We assayed this material biophysically and in crystallization assays and obtained a different crystal form of mature TGFß2, which adopted a conformation deviating from previous structures, with a distinct dimeric conformation that would require significant rearrangement for binding of TGFß receptors. This new conformation may be reversibly adopted by a certain fraction of the mature TGß2 population and represent a hitherto undescribed additional level of activity regulation of the mature growth factor once the latency-associated protein has been separated.

Identification of a ligand binding hot spot and structural motifs replicating aspects of tyrosyl-DNA phosphodiesterase I (TDP1) phosphoryl recognition by crystallographic fragment cocktail screening.

Tyrosyl DNA-phosphodiesterase I (TDP1) repairs type IB topoisomerase (TOP1) cleavage complexes generated by TOP1 inhibitors commonly used as anticancer agents. TDP1 also removes DNA 3' end blocking lesions generated by chain-terminating nucleosides and alkylating agents, and base oxidation both in the nuclear and mitochondrial genomes. Combination therapy with TDP1 inhibitors is proposed to synergize with topoisomerase targeting drugs to enhance selectivity against cancer cells exhibiting deficiencies in parallel DNA repair pathways. A crystallographic fragment screening campaign against the catalytic domain of TDP1 was conducted to identify new lead compounds. Crystal structures revealed two fragments that bind to the TDP1 active site and exhibit inhibitory activity against TDP1. These fragments occupy a similar position in the TDP1 active site as seen in prior crystal structures of TDP1 with bound vanadate, a transition state mimic. Using structural insights into fragment binding, several fragment derivatives have been prepared and evaluated in biochemical assays. These results demonstrate that fragment-based methods can be a highly feasible approach toward the discovery of small-molecule chemical scaffolds to target TDP1, and for the first time, we provide co-crystal structures of small molecule inhibitors bound to TDP1, which could serve for the rational development of medicinal TDP1 inhibitors.

Purification of Recombinant DHHC Proteins Using an Insect Cell Expression System.

DHHC enzymes are a family of integral membrane proteins that catalyze the posttranslational addition of palmitate, a 16-carbon fatty acid, onto a cysteine residue of a protein. While the library of identified palmitoylated proteins has grown tremendously over the years, biochemical and mechanistic studies on DHHC proteins are challenged by the innate difficulty of purifying the enzyme in large amounts. Here we describe our protocol for preparing recombinant DHHC proteins tagged with a hexahistidine sequence and a FLAG epitope that aid in the purification. This procedure has been tested successfully in purifying several members of the enzyme family; DHHC3 and its catalytically inactive cysteine mutant, DHHS3 are used as examples. The recombinant protein is extracted from whole cell lysates using the detergent dodecylmaltoside (DDM) and is subjected to a two-column purification. Homogeneity and monodispersity of the purified protein are checked by size exclusion chromatography (SEC). A preparation from a 400-mL infection of Sf9 insect cell culture typically yields 0.5 mg of DHHC3 and 1.0 mg of catalytically inactive DHHS3. Both forms appear monodisperse up to a concentration of 1 mg/mL by SEC.

Recombinant protein purification in baculovirus-infected Rachiplusia nu larvae: An approach towards a rational design of downstream processing strategies based on chromatographic behavior of proteins.

Expression of recombinant proteins with baculovirus-infected insect larvae is a scarcely investigated alternative in comparison to that in insect cell lines, a system with growing popularity in the field of biotechnology. The aim of this study was to investigate the chromatographic behavior and physicochemical properties of the proteome of Rachiplusia nu larvae infected with recombinant Autographa californica multiple nucleopolyhedrosis virus (AcMNPV), in order to design rational purification strategies for the expression of heterologous proteins in this very complex and little-known system, based on the differential absorption between target recombinant proteins and the system's contaminating ones. Two-dimensional (2D) gel electrophoresis showed differences in the protein patterns of infected and non-infected larvae. Hydrophobic interaction matrices adsorbed the bulk of larval proteins, thus suggesting that such matrices are inappropriate for this system. Only 0.03% and 2.9% of the total soluble protein from the infected larval extract was adsorbed to CM-Sepharose and SP-Sepharose matrices, respectively. Immobilized metal ion affinity chromatography represented a solid alternative because it bound only 1.4% of the total protein, but would increase the cost of the purification process. We concluded that cation-exchange chromatography is the best choice for easy purification of high-isoelectric-point proteins and proteins with arginine tags, since very few contaminating proteins co-eluted with our target protein.

Carboxymethylated polyethylenimine modified magnetic nanoparticles specifically for purification of His-tagged protein.

Employing immobilized metal-ion affinity chromatography and magnetic separation could ideally provide a useful analytical strategy for purifying His-tagged protein. In the current study, a facile route was designed to prepare CMPEI-Ni2+ @SiO2 @Fe3 O4 (CMPEI=carboxymethylated polyethyleneimine) magnetic nanoparticles composed of a strong magnetic core of Fe3 O4 and a Ni2+ -immobilized carboxymethylated polyethyleneimine coated outside shell, which was formed by electrostatic interactions between polyanionic electrolyte of carboxymethylated polyethyleneimine and positively charged surface of 3-(trimethoxysilyl)propylamin modified SiO2 @Fe3 O4 . The resulting CMPEI-Ni2+ @SiO2 @Fe3 O4 composite nanoparticles displayed well-uniform structure and high magnetic responsiveness. Hexa His-tagged peptides and purified His-tagged recombinant retinoid X receptor alpha were chosen as the model samples to evaluate the adsorption, capacity, and reusability of the composite nanoparticles. The results demonstrated the CMPEI-Ni2+ @SiO2 @Fe3 O4 nanoparticles possessed rapid adsorption, large capacity, and good recyclability. The obtained nanoparticles were further used to purify His-tagged protein in practical environment. It was found that the nanoparticles could selectively capture His-tagged recombinant retinoid X receptor protein from complex cell lysate. Owing to its easy synthesis, large binding capacity, and good reusability, the prepared CMPEI-Ni2+ @SiO2 @Fe3 O4 magnetic nanoparticles have great potential for application in biotechnological fields.

Biosynthesis and isolation of selenoneine from genetically modified fission yeast.

Selenoneine, a naturally occurring form of selenium, is the selenium analogue of ergothioneine, a sulfur species with health relevance not only as a purported antioxidant but likely also beyond. Selenoneine has been speculated to exhibit similar effects. To study selenoneine's health properties as well as its metabolic transformation, the pure compound is required. Chemical synthesis of selenoneine, however, is challenging and biosynthetic approaches have been sought. We herein report the biosynthesis and isolation of selenoneine from genetically modified fission yeast Schizosaccharomyces pombe grown in a medium containing sodium selenate. After cell lysis and extraction with methanol, selenoneine was purified by three consecutive preparative reversed-phase HPLC steps. The product obtained at the mg level was characterised by high resolution mass spectrometry, NMR and HPLC/ICPMS. Biosynthesis was found to be a promising alternative to chemical synthesis, and should be suitable for upscaling to produce higher amounts of this important selenium species in the future.

Systematic analysis of the expression, solubility and purification of a passenger protein in fusion with different tags.

Purification of recombinant proteins is often achieved using a purification tag which can be located either at the N- or C-terminus of a passenger protein of interest. Many purification tags exist and their advantages and limitations are well documented. However, designing fusion proteins can be a challenging task to get a fully expressed, soluble and highly purified passenger protein. Besides, there is a lack of systematic studies on the use of a single tag versus combined tags and on the effect of the position of the tags in the construct. In the present study, 9 different fusion proteins were expressed in Escherichia coli using some of the most commonly used purification tags: maltose-binding protein (MBP), glutathione S-transferase (GST) and polyHis tag. The expression and purification of N-terminus single-tagged fusion proteins (MBP, GST and polyHis) and fusion proteins with combined tags at different positions have been tested. Both the identity of the tag(s) and its position were found to have a strong effect on the expression, solubility and purification yields of the fusion proteins. Consequently, the different fusion proteins assayed have shown varying expression, solubility and purification yields, which were also dependent on the passenger protein. Therefore, there is a compelling need to design various fusion proteins with different single or combined tags to identify optimized constructions allowing to achieve high levels of expression, solubility and purification of the passenger protein.

Chitosan/cellulose-based beads for the affinity purification of histidine-tagged proteins.

Chitosan/cellulose-based beads (CCBs) for the affinity purification of histidine-tagged proteins were prepared from chitosan/cellulose dissolved in ionic liquid as a solvent, and their structures were characterized by Fourier transform infrared spectroscopy, transmission electron microscopy, and thermogravimetric analysis. The affinity purification was used to separate hexahistidine-tagged (his-tagged) enhanced green fluorescent protein (EGFP) from Escherichia coli. The results showed that Zn2+-CCB exhibited more specific adsorption capacity toward the target protein compared with Ni2+-CCB and Cu2+-CCB. The maximum adsorption of EGFP was 1.84 mg/g of Zn2+-CCB, with 90% purity under the optimized conditions (ionic strength (1.0 M NaCl), pH (7.2) and imidazole concentration (500 mM)). In addition, a regeneration method for the sorbent was further developed by washing with ethylenediaminetetraacetic acid disodium and then reimmobilizing with metal ions. This technique is an alternative method for the purification of his-tagged proteins, making the process more economical, fast, stable, and large batch.

High-level expression of soluble recombinant proteins in Escherichia coli using an HE-maltotriose-binding protein fusion tag.

Recombinant proteins are commonly expressed in prokaryotic expression systems for large-scale production. The use of genetically engineered affinity and solubility enhancing fusion proteins has increased greatly in recent years, and there now exists a considerable repertoire of these that can be used to enhance the expression, stability, solubility, folding, and purification of their fusion partner. Here, a modified histidine tag (HE) used as an affinity tag was employed together with a truncated maltotriose-binding protein (MBP; consisting of residues 59-433) from Pyrococcus furiosus as a solubility enhancing tag accompanying a tobacco etch virus protease-recognition site for protein expression and purification in Escherichia coli. Various proteins tagged at the N-terminus with HE-MBP(Pyr) were expressed in E. coli BL21(DE3) cells to determine expression and solubility relative to those tagged with His6-MBP or His6-MBP(Pyr). Furthermore, four HE-MBP(Pyr)-fused proteins were purified by immobilized metal affinity chromatography to assess the affinity of HE with immobilized Ni2+. Our results showed that HE-MBP(Pyr) represents an attractive fusion protein allowing high levels of soluble expression and purification of recombinant protein in E. coli.

Chitosan-nickel film based interferometric optical fiber sensor for label-free detection of histidine tagged proteins.

An interferometric fiber sensor for detection of hexa-histidine tagged microcin (His-MccS) is reported and experimentally demonstrated. This intermodal fiber sensor is implemented by a no-core fiber (NCF) functionalized with chitosan (CS)-nickel (Ni) film for direct detection of small peptide: microcin. The fiber intermodal sensor relies on the refractive index modulations due to selective adsorption event at the chitosan (CS)-nickel (Ni) film. Owing to the strong affinity between Ni2+ ions and histidine, the immobilized Ni2+ ions in the chitosan film were utilized as binding agents for the direct detection of hexa-histidine tagged microcin. A comparative study in relation to different target size was conducted: full proteins trypsin, bovine serum albumin (BSA) and human serum albumin (HSA), with high histidine content on their surface and His-MccS (peptide, 11.6kDa), have been employed for sensor evaluation. Results have shown selectivity for His-MccS relative to trypsin, BSA and HSA. The most telling contribution of this study is the fast detection of small biomolecule His-MccS compared to standard detection procedures like SDS-PAGE and western blot. The proposed sensor exhibits His-MccS detection sensitivity of 0.0308nm/(ng/ml) in the range of (0-78) ng/ml with concentration detection limit of 0.8368ng/ml.

Screening Fusion Tags for Improved Recombinant Protein Expression in E. coli with the Expresso® Solubility and Expression Screening System.

The simplicity, speed, and low cost of bacterial culture make E. coli the system of choice for most initial trials of recombinant protein expression. However, many heterologous proteins are either poorly expressed in bacteria, or are produced as incorrectly folded, insoluble aggregates that lack the activity of the native protein. In many cases, fusion to a partner protein can allow for improved expression and/or solubility of a difficult target protein. Although several different fusion partners have gained favor, none are universally effective, and identifying the one that best improves soluble expression of a given target protein is an empirical process. This unit presents a strategy for parallel screening of fusion partners for enhanced expression or solubility. The Expresso® Solubility and Expression Screening System includes a panel of seven distinct fusion partners and utilizes an extremely simple cloning strategy to enable rapid screening and identification of the most effective fusion partner. © 2017 by John Wiley & Sons, Inc.

Enhanced expression of lipase I from Galactomyces geotrichum by codon optimisation in Pichia pastoris.

Relatively poor heterologous protein yields have limited the commerical applications of Galactomyces geotrichum lipase I (GGl I) efficacy trials. To address this, we have redesigned the GGl I gene to preferentially match codon frequencies of Pichia pastoris (P. pastoris) while retaining the same amino acid sequence. The wild type and codon optimised GGl I (GGl I-wt and GGl I-op) were synthesised and cloned into pPICZαA with an N-terminal 6 × His tag sequence and expressed in P. pastoris X 33. The hydrolytic activity of GGl I-op was 150 U/mL, whereas the activity of the GGl I-wt could not be detected. GGl I-op recombinant proteins were purified by Ni-affinity chromatography and then characterised. The identity and purity of GGl I were confirmed by SDS-PAGE, MALDI-TOF mass spectrometry and Western blot analysis. Enzymatic deglycosylation was used to show that the lipase is a glycosylated protein, containing ∼10% sugar. The molecular weight (MW) of the GGl I secreted by recombinant P. pastoris was approximated at 63 kDa. The optimum pH and temperature of the recombinant lipase were 8.0 and 35 °C, respectively. The enzyme was active over a broad pH range (7.0-9.0) and temperature range (20 °C-45 °C). The lipase showed high activity toward medium- and long-chain fatty acid methyl esters (C8-C16) and retained much of its activity in the presence of Tween-80 and Trition X-100. Lipase activity was stimulated by Mg2+, Ca2+, Mn2+ and Cu2+ and inhibited by Fe2+, Fe3+, Zn2+ and Co2+. This lipase may prove useful to the detergent industry and in organic synthesis reactions.

A structure based plasma protein pre-fractionation using conjoint immobilized metal/chelate affinity (IMA) system.

The potential of immobilized metal/chelate affinity (IMA) in a continuous fashion, referred as conjoint approach, to pre-fractionate plasma proteins (in their native state) prior to LC-MS analysis was investigated in this study. Four transition metal-ions (Co (II), Zn (II), Ni (II) and Cu (II)) were individually chelated with IDA (iminodiacetic acid) coated CIM (Convective Interaction Media) disks and placed in a single housing in the following sequential order: IDA-Co (II)→IDA-Zn (II)→IDA-Ni (II)→IDA-Cu (II). The rationale behind this order is to retain proteins based on their specific requirement for surface exposed histidine topography. This structural pre-fractionation hypothesis was successfully proven using four human plasma proteins (fibrinogen, IgG, transferrin, and albumin) with varying histidine topographies. This conjoint IMA pre-fractionation strategy not only fractionated proteins (from plasma) based on their native surface histidine topography, but also identified 157 proteins from human plasma. The advantage of our conjoint IMA is its ability to fractionate proteins in their native state and reduce plasma complexity in a single step by employing single buffer system.

Expression and purification of tau protein and its frontotemporal dementia variants using a cleavable histidine tag.

Recombinant tau protein is widely used to study the biochemical, cellular and pathological aspects of tauopathies, including Alzheimer's disease and frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTPD-17). Pure tau in high yield is a requirement for in vitro evaluation of the protein's physiological and toxic functions. However, the preparation of recombinant tau is complicated by the protein's propensity to aggregate and form truncation products, necessitating the use of multiple, time-consuming purification methods. In this study, we investigated parameters that influence the expression of wild type and FTPD-17 pathogenic tau, in an attempt to identify ways to maximise expression yield. Here, we report on the influence of the choice of host strain, induction temperature, duration of induction, and media supplementation with glucose on tau expression in Escherichia coli. We also describe a straightforward process to purify the expressed tau proteins using immobilised metal affinity chromatography, with favourable yields over previous reports. An advantage of the described method is that it enables high yield production of functional oligomeric and monomeric tau, both of which can be used to study the biochemical, physiological and toxic properties of the protein.

Recombinant production, purification and characterization of vessel dilator in E. coli.

Vessel dilator is a 3.9-KDa potent anticancer peptide and a valuable candidate in the treatment of conditions such as congestive heart failure and acute renal failure amongst others. Here we report the recombinant production of vessel dilator in Escherichia coli. Three different synthetic ORF's dubbed VDI, VDII and VDIII, each encoding a trimmer of the vessel dilator peptide attached to a His tag sequence at their C- terminal, were synthesized and placed in pET21c expression vectors. The highest yield, following expression in E. coli BL21 (DE3), was recorded with VDII that carried the shortest fusion partner. Subsequent to the initial capture of the fusion protein by a Ni affinity column, the vessel dilator monomers were cleaved by trypsin treatment, and further purified to at least 90% homogeneity by anion exchange chromatography. De-novo sequencing and in vivo anticancer activity tests were used to verify the peptide sequence and its biological activity, respectively. The final yield was estimated to be approximately 15 mg of the purified vessel dilator per gram wet weight of the bacterial cells.

A non-cleavable hexahistidine affinity tag at the carboxyl-terminus of the HIV-1 Pr55Gag polyprotein alters nucleic acid binding properties.

HIV Gag (Pr55Gag), a multidomain polyprotein that orchestrates the assembly and release of the human immunodeficiency virus (HIV), is an active target of antiretroviral inhibitor development. However, highly pure, stable, recombinant Pr55Gag has been difficult to produce in quantities sufficient for biophysical studies due to its susceptibility to proteolysis by cellular proteases during purification. Stability has been improved by using a construct that omits the p6 domain (Δp6). In vivo, p6 is crucial to the budding process and interacts with protein complexes in the ESCRT (Endosomal Sorting Complexes Required for Transport) pathway, it has been difficult to study its role in the context of Gag using in vitro approaches. Here we report the generation of a full length Gag construct containing a tobacco etch virus (TEV)-cleavable C-terminal hexahistidine tag, allowing a detailed comparison of its nucleic acid binding properties with other constructs, including untagged, Δp6, and C-terminally tagged (TEV-cleavable and non-cleavable) Gags, respectively. We have developed a standard expression and purification protocol that minimizes nucleic acid contamination and produces milligram quantities of full length Gag for in vitro studies and compound screening purposes. We found that the presence of a carboxyl-terminal hexahistidine tag changes the nucleic binding properties compared to the proteins that did not contain the tag (full length protein that was either untagged or reulted from removal of the tag during purification). The HIV Gag expression and purification protocol described herein provides a facile method of obtaining large quantities of high quality protein for investigators who wish to study the full length protein or the effect of the p6 domain on the biophysical properties of Gag.

Liquid chromatography-electrospray ionization mass spectrometry for the simultaneous quantitation of collagen and elastin crosslinks.

We have developed a novel chromatographic analytical method for the simultaneous quantitation of collagen crosslinks. Seven non-derivatised crosslinks could be separated on a Cogent Diamond Hydride HPLC column using either isocratic or gradient conditions then detected by mass spectrometry. The total run time was less than 10min which is significantly shorter than that previously reported. This is the first method in which histidinohydroxylysinonorleucine (HHL) and histidinohydroxymero-desmosine (HHMD) were separated and identified by mass spectrometry without the need for pre- or post-column derivatization. The CVs of the retention times of all seven crosslinks were less than 1% and the limit of detection (LOD) and the limits of quantitation (LOQ) were 0.07-0.13pmol/µL and 0.20-0.38pmol/µL, respectively. This novel method was used for the routine analysis and quantitation of crosslinks in different animal skins in which potential new collagen crosslinks were identified that are as yet undocumented.

Preparation of core-shell structure Fe3 O4 @SiO2 superparamagnetic microspheres immoblized with iminodiacetic acid as immobilized metal ion affinity adsorbents for His-tag protein purification.

The core-shell structure Fe3 O4 /SiO2 magnetic microspheres were prepared by a sol-gel method, and immobiled with iminodiacetic acid (IDA) as metal ion affinity ligands for protein adsorption. The size, morphology, magnetic properties and surface modification of magnetic silica nanospheres were characterized by various modern analytical instruments. It was shown that the magnetic silica nanospheres exhibited superparamagnetism with saturation magnetization values of up to 58.1 emu/g. Three divalent metal ions, Cu(2+) , Ni(2+) and Zn(2+) , were chelated on the Fe3 O4 @SiO2 -IDA magnetic microspheres to adsorb lysozyme. The results indicated that Ni(2+) -chelating magnetic microspheres had the maximum adsorption capacity for lysozyme of 51.0 mg/g, adsorption equilibrium could be achieved within 60 min and the adsorbed protein could be easily eluted. Furthermore, the synthesized Fe3 O4 @SiO2 -IDA-Ni(2+) magnetic microspheres were successfully applied for selective enrichment lysozyme from egg white and His-tag recombinant Homer 1a from the inclusion extraction expressed in Escherichia coli. The result indicated that the magnetic microspheres showed unique characteristics of high selective separation behavior of protein mixture, low nonspecific adsorption, and easy handling. This demonstrates that the magnetic silica microspheres can be used efficiently in protein separation or purification and show great potential in the pretreatment of the biological sample.

Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli.

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein belonging to the prion family. A chromatographic method for the purification of (His)6-tagged recombinant Shadoo expressed as inclusion bodies is described. The inclusion bodies are solubilized in 8 M urea and bound to a Ni(2+)-charged column to perform ion affinity chromatography. Bound proteins are eluted by a gradient of imidazole. Fractions containing Shadoo protein are subjected to size exclusion chromatography to obtain a highly purified protein. In the final step purified Shadoo is desalted to remove salts, urea and imidazole. Recombinant Shadoo protein is an important reagent for biophysical and biochemical studies of protein conformation disorders occurring in prion diseases. Many reports demonstrated that prion neurodegenerative diseases originate from the deposition of stable, ordered amyloid fibrils. Sample protocols describing how to fibrillate Shadoo into amyloid fibrils at acidic and neutral/basic pHs are presented. The methods on how to produce and fibrillate Shadoo can facilitate research in laboratories working on prion diseases, since it allows for production of large amounts of protein in a rapid and low cost manner.