His-tagged Protein Research Articles

NTA-Cholesterol Analogue for the Nongenetic Liquid-Ordered Phase-Specific Functionalization of Lipid Membranes with Proteins.

The nongenetic modification of cell membranes with proteins is a straightforward way of cellular engineering. In these processes, it is important to specifically address the proteins to liquid-ordered (Lo) or liquid-disordered (Ld) domains as this can largely affect their biological functions. Herein, we report a cholesterol analogue (CHIM) with a nitrilotriacetic acid (NTA) headgroup, named CHIM-NTA. CHIM-NTA integrates into lipid membranes similar to the widely used phospholipid-derived DGS-NTA and, when loaded with Ni2+, allows for specific membrane immobilization of any polyhistidine-tagged proteins of choice. Yet, unlike DGS-NTA, it localizes to the Lo phase in phase-separated giant unilamellar vesicles (GUVs) and allows addressing His-tagged proteins to Lo domains. Furthermore, CHIM-NTA readily integrates into the membranes of live cells and thus enables the nongenetic modification of the cell surface with proteins. Overall, CHIM-NTA provides a facile and flexible way to modify biological membranes, in particular Lo domains, with His-tagged proteins and can serve as a broadly applicable molecular tool for cell surface engineering.

Micropatterning of Quantum Dots for Biofunctionalization and Nanoimaging

Micron-scale patterning of colloidal quantum dots (QDs) is extremely important for the fabrication of high-performance Quantum dot Light-Emitting Diode (QLED) displays, biosensing, and super-resolution imaging. Thus, several nondestructive methods have been recently proposed, such as spatial self-organization. However, none of them can be useful for biofunctionalization or nanoimaging. To address this limitation, we propose a method to create micropatterns of QDs of any shape and size. UV photolithography assisted by a digital micromirror device (DMD) and silanization allow creating an adhesive layer, on which QDs micropatterns can be assembled with a 2 μm resolution. The patterns are composed of a monolayer of CdSe/CdS/CdZnS/ZnS core/multishell QDs (7 ± 1 nm in diameter, emitting at 590 nm) with a high surface density (typically 4000 QDs/μm2). We also demonstrate that it is possible to reversibly bind any kind of His-Tagged proteins on the QDs surface. This is highlighted by measuring FRET (Förster Resonance Energy Transfer) with a dedicated polymer exhibiting on one end Alexa Fluor 647 (AF647) and on the other end eight imidazole cycles, allowing chelation on the quantum dots’ surface. Therefore, this patterning protocol provides a path to combine nanoimaging with cell patterning through a relevant biofunctionalization.

Development of HEK293T-produced recombinant receptor-Fc proteins as potential candidates against canine distemper virus.

Canine distemper (CD) is a highly contagious viral disease worldwide. Although live attenuated vaccine is available as a preventive measure against the disease, cases of vaccination failure highlight the importance of potential alternative agent against canine distemper virus (CDV). CDV infects cells mainly by binding signaling lymphocyte activation molecule (SLAM) and Nectin-4 receptor. Here, to develop a new and safe antiviral biological agent for CD, we constructed and expressed CDV receptor proteins fused with Fc region of canine IgG-B, namely, SLAM-Fc, Nectin-Fc and SLAM-Nectin-Fc in HEK293T cells, and antiviral activity of these receptor-Fc proteins was subsequently evaluated. The results showed that the receptor-Fc proteins efficiently bound to receptor binding domain (RBD) of CDV-H, meanwhile, these receptor-Fc proteins competitively inhibited the binding of His-tagged receptor proteins (SLAM-His or Nectin-His) to CDV-H-RBD-Flag protein. Importantly, receptor-Fc proteins exhibited potent anti-CDV activity in vitro. Treatment with receptor-Fc proteins at the pre-entry stage dramatically suppressed CDV infectivity in Vero cells stably expressing canine SLAM. The minimum effective concentration (MEC) of SLAM-Fc, Nectin-Fc and SLAM-Nectin-Fc was 0.2 μg/mL, 0.2 μg/mL, 0.02 μg/mL. The 50% inhibition concentration (IC50) of three proteins was 0.58 μg/mL, 0.32 μg/mL and 0.18 μg/mL, respectively. Moreover, treatment with receptor-Fc proteins post viral infection can also inhibit CDV reproduction, the MEC of SLAM-Fc, Nectin-Fc and SLAM-Nectin-Fc was same as pre-treatment, and the IC50 of receptor-Fc proteins was 1.10 μg/mL, 0.99 μg/mL and 0.32 μg/mL, respectively. The results suggested that the receptor-Fc proteins were more effective for pre-entry treatment than post-infection treatment, furthermore, SLAM-Nectin-Fc was more effective than SLAM-Fc and Nectin-Fc. These findings revealed the receptor-Fc proteins were promising candidates as inhibitor against CDV.

Efficient Purification of Polyhistidine-Tagged Recombinant Proteins Using Functionalized Corundum Particles.

Immobilized metal affinity chromatography (IMAC) is a popular and valuable method for the affinity purification of polyhistidine-tagged recombinant proteins. However, it often shows practical limitations, which might require cumbersome optimizations, additional polishing, and enrichment steps. Here, we present functionalized corundum particles for the efficient, economical, and fast purification of recombinant proteins in a column-free format. The corundum surface is first derivatized with the amino silane APTES, then EDTA dianhydride, and subsequently loaded with nickel ions. The Kaiser test, well known in solid-phase peptide synthesis, was used to monitor amino silanization and the reaction with EDTA dianhydride. In addition, ICP-MS was performed to quantify the metal-binding capacity. His-tagged protein A/G (PAG), mixed with bovine serum albumin (BSA), was used as a test system. The PAG binding capacity was around 3 mg protein per gram of corundum or 2.4 mg per 1 mL of corundum suspension. Cytoplasm obtained from different E. coli strains was examined as examples of a complex matrix. The imidazole concentration was varied in the loading and washing buffers. As expected, higher imidazole concentrations during loading are usually beneficial when higher purities are desired. Even when higher sample volumes, such as one liter, were used, recombinant protein down to a concentration of 1 µg/mL could be isolated selectively. Comparing the corundum material with standard Ni-NTA agarose beads indicated higher purities of proteins isolated using corundum. His6-MBP-mSA2, a fusion protein consisting of monomeric streptavidin and maltose-binding protein in the cytoplasm of E. coli, was purified successfully. To show that this method is also suitable for mammalian cell culture supernatants, purification of the SARS-CoV-2-S-RBD-His8 expressed in human Expi293F cells was performed. The material cost of the nickel-loaded corundum material (without regeneration) is estimated to be less than 30 cents for 1 g of functionalized support or 10 cents per milligram of isolated protein. Another advantage of the novel system is the corundum particles' extremely high physical and chemical stability. The new material should be applicable in small laboratories and large-scale industrial applications. In summary, we could show that this new material is an efficient, robust, and cost-effective purification platform for the purification of His-tagged proteins, even in challenging, complex matrices and large sample volumes of low product concentration.

IL-13 protects epithelial cells from SARS-CoV-2 infection by inhibiting early ACE2-mediated events

Abstract Severe Acute Respiratory Syndrome coronavirus-2 (SARS-CoV-2) infection is mediated by binding of viral spike (S) protein to membrane ACE2 on host epithelial cells, preferentially in the airway. Asthmatics with type 2 (T2) inflammation are reportedly less susceptible to disease, but the underlying mechanisms are unclear. We recently showed that ACE2expression is decreased in airway epithelial cells from T2 asthmatics and is consistently suppressed in human primary epithelial cells cultured with IL-13 (PMID: 32422146). To dissect IL-13/SARS-CoV-2 interactions, we assessed the effects of IL-13 on discrete events in SARS-CoV-2 infection. S1 protein binding to green monkey kidney Vero E6, human lung Calu-3 epithelial cells and HEK293T cells stably transfected with human ACE2(hACE2-HEK293T) was measured by flow cytometry with His-tagged S1 protein. S protein-mediated viral entry into Vero E6 and hACE2-HEK293T cells was assessed using S protein-pseudotyped lentiviral particles carrying a GFP reporter. ACE2transcription in Vero E6, Calu-3 and primary human airway epithelial cells was measured by RT-qPCR. ACE2 expression on Vero E6, Calu-3 and hACE2-HTK293T cells was evaluated by flow cytometry. USA-WA1/2020 SARS-CoV-2 was used to infect Calu-3 cells. IL-13 strongly inhibited S protein binding, cell entry, and infection of epithelial cells by SARS-CoV-2. These effects resulted at least in part from IL-13-dependent inhibition of ACE2expression because IL-13 did not affect S protein binding to, and S protein-pseudotyped lentivirus entry into, hACE2-HEK293T cells that do not downregulate ACE2 in response to IL-13. We suggest that IL-13 protects epithelial cells from SARS-CoV-2 infection by inhibiting early ACE2-mediated events. Supported by grant from NIAID: U19 AI125357

Analysis of histidine-tagged recombinant proteins from nickel and copper coated surfaces by direct electrospray ionization and desorption electrospray ionization mass spectrometry.

Purification of recombinant proteins is a necessary step for functional or structural studies and other applications. Immobilized metal affinity chromatography is a common recombinant protein purification method. Mass spectrometry (MS) allows for confirmation of identity of expressed proteins and unambiguous detection of enzymatic substrates and reaction products. We demonstrate the detection of enzymes purified on immobilized metal affinity surfaces by direct or ambient ionization MS, and follow their enzymatic reactions by direct electrospray ionization (ESI) or desorption electrospray ionization (DESI). A protein standard, His-Ubq, and two recombinant proteins, His-SHAN and His-CS, expressed in Escherichia coli were immobilized on two immobilized metal affinity systems, Cu-nitriloacetic acid (Cu-NTA) and Ni-NTA. The proteins were purified on surface, and released in the ESI spray solvent for direct infusion, when using the 96-well plate form factor, or analyzed directly from immobilized metal affinity-coated microscope slides by DESI-MS. Enzyme activity was followed by incubating the substrates in wells or by depositing substrate on immobilized protein on coated slides for analysis. Small proteins (His-Ubq) and medium proteins (His-SAHN) could readily be detected from 96-well plates by direct infusion ESI, or from microscope slides by DESI-MS after purification on surface from clarified E. coli cell lysate. Protein oxidation was observed for immobilized proteins on both Cu-NTA and Ni-NTA; however, this did not hamper the enzymatic reactions of these proteins. Both the nucleosidase reaction products for His-SAHN and the methylation product of His-CS (theobromine to caffeine) were detected. The immobilization, purification, release and detection of His-tagged recombinant proteins using immobilized metal affinity surfaces for direct infusion ESI-MS or ambient DESI-MS analyses were successfully demonstrated. Recombinant proteins were purified to allow identification directly out of clarified cell lysate. Biological activities of the recombinant proteins were preserved allowing the investigation of enzymatic activity via MS.

Abstract 3457: Characterization of acquired resistance mutations to menin inhibitors

Abstract The menin-MLL1 interaction is critical for development of acute leukemias driven by MLL1 rearrangements (MLLr) or mutations in the Nucleophosmin 1 gene (NPM1c). Inhibition of the menin-MLL1 interaction by SNDX-5613 (revumenib) has demonstrated robust clinical responses in the current AUGMENT clinical trial (NCT04065399). During the trial, some responders relapsed during treatment due to acquired resistance in MEN1. Somatic MEN1 mutations were found at residues M327, G331 or T349 which diminished SDNX-5613 binding affinity and mediated therapeutic resistance. The presence of acquired resistance validates MEN1 as a therapeutic target in MLLr and NPM1c AML patients. Here we characterize the effects of these mutations on the activity of 6 menin inhibitor chemotypes currently in clinical trials (NCT04065399/Syndax, NCT04067336/Kura, NCT04811560/JNJ, NCT04988555/Sumitomo, NCT04752163/Daiichi, NCT05153330/Biomea). In vitro activity and binding modes for these compounds were evaluated in wild-type (WT) and mutant menins using (i) competition binding assays, (ii) cell-based proliferation assays and (iii) X-ray co-crystallography. For binding, His6-tagged MEN1 mutant proteins (G331R, M327I, M327V, T349M) were expressed and purified. Binding affinities were measured in competition binding format. The menin-MLL interaction was monitored by HTRF using Terbium labeled anti-His6 antibody and FITC labeled MLL peptide (4-43). Acquired mutations affected binding affinities (Ki) to varying degrees. Notably, M327I/V mutations reduced binding for all menin-MLL inhibitors ranging from ~30-300, indicating a class effect for this mutation. An irreversible Biomea chemotype did not inhibit menin-MLL binding in our assays. The decreased binding affinity to M327I was reflected in cell-based proliferation assays. Menin mutations were introduced into MV4;11 cells using CRISPR-Cas9 in conjunction with a homology directed repair template to edit the endogenous MEN1 coding sequence. Clonal lines were established harboring homozygous (homo) and heterozygous (het) M327I mutations. The M327I (het) MV4;11 cells experienced 15-50-fold shifts in IC50 vs WT cells, consistent with the reduced binding affinities. The molecular basis for sensitivity to M327 acquired resistance was examined by X-ray co-crystallography of inhibitors bound to M327I and WT menin. Both KO-539 and SNDX-5613 show notable changes in binding in M327I vs WT menin. The isoleucine creates a steric clash, displacing their position in the pocket as previously noted. The Janssen chemotype shows a novel binding mode. Although it has 30-fold lower affinity for M327I, it shows little change in its bound position to M327I menin. Given the clinical validation of menin inhibition in AML, the design of next generation compounds that block MLL1 binding while avoiding acquired MEN1 mutations may be a strategy to overcome acquired resistance to first generation menin inhibitors. Citation Format: Florian Perner, Sheng F. Cai, Daniela V. Wenge, Jeonghyeon Kim, Jevon Cutler, Radosław P. Nowak, Joel Cassel, Shivendra Singh, Shipra Bijpuria, William H. Miller, Eytan M. Stein, Ross L. Levine, Eric S. Fischer, Gerard M. McGeehan, Scott A. Armstrong. Characterization of acquired resistance mutations to menin inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3457.

Prokaryotic Expression and Enzymatic Properties of Lipase from Schizochytrium Pombe

Based on the codon preference of Escherichia coli, the gene of lipase from Schizochytrium pombe was optimized and the codon adaptation index was 0.8 and the gene was expressed in Escherichia coli as a His-tag fusion protein using pET-30a as the expression vector. The recombinant strain with OD600 about 0.6 was induced with 1 mmol/L isopropyl-β-D-thiogalactoside at 30 °C for 12 h. The purified recombinant lipase STGL3 had a molecular mass of 32 kDa, and the purified lipase had a specific enzyme activity of 2.29 μmol/(min•mg), which was 2.3 times higher than before, and a protein content of 650.00 mg/L. The recombinant STGL3 showed the highest hydrolysis activity at 40 °C and pH 7.5, respectively. The effect of different metal ion concentrations on enzyme activity varied, lipase STGL3 was stable in the presence of Ca2+, while Mg2+, Mn2+, Zn2+, Fe2+ and EDTA had an inhibitory effects on lipase activity. Different types of surfactants and organic solvents inhibited the enzymatic activity of recombinant lipase to different extents. The recombinant lipases showed a preference towards pNP-esters with medium and longer acyl-chains. (C12, C14 and C16) and the recombinant lipases showed relatively weak lipase activity towards the shorter carbon chains. The kinetic constants of lipase were 0.29 mmol/L for Km, 2.28 mmol/(L•min-1 ) for Vmax, and 6.19 S-1 for Kcat. The present study not only achieved the expression and characterization of enzymatic properties of lipase from Leptosphaeria japonica, but also provided some basis for the study of enhancing the quality and quality of algal oil by means of genetic engineering, and further exploring the potential industrial applications of Leptosphaeria japonica lipase.

Selective Immobilization of His-Tagged Enzyme on Ni-Chelated Ion Exchange Resin and Its Application in Protein Purification.

Ion exchange resins are suitable as carriers for immobilized enzymes because of their stable physicochemical properties, appropriate particle size and pore structure, and lower loss in continuous operation. In this paper, we report the application of the Ni-chelated ion exchange resin in the immobilization of His-tagged enzyme and protein purification. Acrylic weak acid cation exchange resin (D113H) was selected from four cationic macroporous resins that could chelate the transition metal ion Ni. The maximum adsorption capacity of Ni was ~198 mg/g. Phosphomannose isomerase (PMI) can be successfully immobilized on Ni-chelated D113H from crude enzyme solution through chelation of transition metal ions with the His-tag on the enzyme. The maximum amount of immobilized PMI on the resin was ~143 mg/g. Notably, the immobilized enzyme showed excellent reusability and maintained 92% of its initial activity with 10 cycles of catalytic reaction. In addition, PMI was successfully purified using an affinity chromatography column prepared by Ni-chelated D113H, which showed the potential for the immobilization and purification process to be realized in one step.

Staphylococcus aureus MazG hydrolyzes oxidized guanine nucleotides and contributes to oxidative stress resistance

We previously reported that knockout of the mazG (SA1292) gene decreases Staphylococcus aureus killing activity against silkworms. S. aureus MazG (SaMazG) has a nucleotide pyrophosphatase domain conserved among MazG family proteins, but its biochemical characteristics are unknown. In the present study, we purified recombinant N-terminal His-tagged SaMazG protein and examined its biochemical activity. SaMazG hydrolyzed GTP, UTP, dGTP, and TTP into nucleoside monophosphates. Hydrolytic activity of SaMazG against ATP, CTP, dATP, and dCTP was low or not detected. SaMazG exhibited high hydrolytic activity against 8-oxo-GTP and 8-oxo-dGTP, oxidized guanine nucleotides, with a Vmax/Km ratio more than 15-fold that of GTP. Furthermore, the S. aureus mazG knockout mutant was sensitive to hydrogen peroxide compared with the parent strain. These results suggest that SaMazG is a nucleotide pyrophosphatase hydrolyzing oxidized guanine nucleotides that contributes to the oxidative stress resistance of S. aureus.

Characterization of Galectin Fusion Proteins with Glycoprotein Affinity Columns and Binding Assays.

Galectins are β-galactosyl-binding proteins that fulfill essential physiological functions. In the biotechnological field, galectins are versatile tools, such as in the development of biomaterial coatings or the early-stage diagnosis of cancer diseases. Recently, we introduced galectin-1 (Gal-1) and galectin-3 (Gal-3) as fusion proteins of a His6-tag, a SNAP-tag, and a fluorescent protein. We characterized their binding in ELISA-type assays and their application in cell-surface binding. In the present study, we have constructed further fusion proteins of galectins with fluorescent protein color code. The fusion proteins of Gal-1, Gal-3, and Gal-8 were purified by affinity chromatography. For this, we have prepared glycoprotein affinity resins based on asialofetuin (ASF) and fetuin and combined this in a two-step purification with Immobilized Metal Affinity chromatography (IMAC) to get pure and active galectins. Purified galectin fractions were analyzed by size-exclusion chromatography. The binding characteristics to ASF of solely His6-tagged galectins and galectin fusion proteins were compared. As an example, we demonstrate a 1.6-3-fold increase in binding efficiency for HSYGal-3 (His6-SNAP-yellow fluorescent protein-Gal-3) compared to the HGal-3 (His6-Gal-3). Our results reveal an apparent higher binding efficiency for galectin SNAP-tag fusion proteins compared to His6-tagged galectins, which are independent of the purification mode. This is also demonstrated by the binding of galectin fusion proteins to extracellular glycoconjugates laminin, fibronectin, and collagen IV. Our results indicate the probable involvement of the SNAP-tag in apparently higher binding signals, which we discuss in this study.

P10 Phage-encoded lysins as promising antibacterials against uropathogenic Escherichia coli

Abstract Background Urinary tract infections (UTIs) represent a major public health issue. Uropathogenic Escherichia coli (UPEC) is a pathogen implicated in the vast majority of UTIs. Although treatable, the rampant and indiscriminate use of antibiotics has led to an alarming emergence of MDR strains. This can lead to complications, treatment failure, and increased rates of mortality and morbidity. Given the prevalence of these strains, we are exploring phage encoded-lysins as potential alternatives to antibiotics. Objectives Our study involved the development of an in silico strategy for the discovery and characterization of prophage-derived lysin sequences targeting the E. coli cell wall and evaluating the antibacterial activity of the recombinant lysins using in vitro assays. Methods Novel lysin sequences were searched by BLAST homology and by screening E. coli prophages in the database (using PHASTER). They were computationally characterized and their domain architecture was examined. Based on the desired physicochemical properties of the 16 lysins, 7 were selected for cloning, expression, and purification as recombinant proteins for the in vitro antibacterial activity. Results In silico analysis showed lysozyme-like domain in 9 out of 16 lysins and the predicted structure was either modular or globular. Cationic and hydrophobic residues at the C-terminal end and cationic residues in the catalytic domains indicate intrinsic bactericidal activity. Lysins (7) were cloned in pET28 vector and purified as His-tagged recombinant proteins by Ni-NTA chromatography. Using spot assay, 3 out of 7 lysins (9–12 μg) showed lysis of E. coli cells (BL21 DE3). The most promising was lysin sequence 5, which showed lysis of E. coli cells, both at stationary as well as mid-log phase cells. Turbidity reduction assay showed a 74.94% drop in OD600 in lysin (15 μM) treated cells after 3 h of incubation, at 37°C. Log killing assay showed 4 log10 reduction in the treated cells. Lysozyme assay demonstrates higher than / comparable lysozyme activity of lysin sequence 5 with the positive control. Conclusions Sixteen novel prophage-encoded lysin sequences have been identified to create a lysin bank (to screen against E. coli), and 7 of them have been purified. Screening shows three lysins having intrinsic bactericidal activity and hence did not require the addition of an Outer Membrane Permeabilizer (OMP). Lysin sequence 5 has exhibited the highest activity against E. coli cells. Enzyme stability and kinetics studies and screening of clinical isolates of E. coli from UTI patients is underway. Investigating this bank of lysins is aimed at the identification and development of a lead ‘enzybiotic’ with desired PK/PD characteristics against UTI-causing E. coli.

Ragweed Major Allergen Amb a 11 Recombinant Production and Clinical Implications

Ragweed pollen is highly allergenic and elicits type I hypersensitivity reactions in the exposed populations. Amb a 11 is a recently discovered component of this pollen, and its biological role in allergy is still being researched. In our study, ragweed allergy patients were recruited prospectively over a three-year period; a comprehensive questionnaire was administered, and sera were collected and stored. The production of recombinant Amb a 11 was achieved in parallel with patients' recruitment. The gene coding for mature protein was inserted in E. coli and in Sf9 Spodoptera frugiperda cells. The recombinant allergens (designated eAmb a 11 and iAmb a 11) were tested for His-tag presence in Western blot. IgE reactivity was evaluated in 150 patients' sera for both recombinant allergen forms in ELISA, with 5 positive sera being tested further by hRBL (humanized rat basophilic leukemia) hexosaminidase release assay. Both allergen forms were proven to be IgE-reactive His-tagged proteins, with an extensive overlap of positive sera (92 toward the former recombinant allergen, 100 toward the latter) and an overall Amb a 11 sensitization prevalence estimated at 68.67%. The hRBL mediator release assay revealed a significant, slightly weaker effect of recombinant allergens when compared with nAmb a 1. Sensitization to this major allergen appears to be associated with more severe asthma symptoms (OR = 4.71, 95% CI = 1.81-12.21). In conclusion, recombinant Amb a 11 is a bona fide allergen, which is IgE-reactive and an inducer of hRBL degranulation. It is an important IgE-reactive component from ragweed pollen, with high IgE sensitization prevalence in the sample population and allergenicity of the recombinant allergen comparable to Amb a 1.

Insight into the Substrate Specificity of Lactobacillus paracasei Aspartate Ammonia-Lyase

Aspartate ammonia-lyase (AAL) catalyzes the reversible conversion reactions of aspartate to fumaric acid and ammonia. In this work, Lactobacillus paracasei LpAAL gene was heterologously expressed in Escherichia coli. As well as a recombinant His-tagged LpAAL protein, a maltose-binding protein (MBP) fused LpAAL protein was used to enhance its protein solubility and expression level. Both recombinant proteins showed broad substrate specificity, catalyzing aspartic acid, fumaric acid, phenylalanine, and tyrosine to produce fumaric acid, aspartic acid, trans-cinnamic acid, and p-coumaric acid, respectively. The optimum reaction pH and temperature of LpAAL protein for four substrates were measured at 8.0 and 40 °C, respectively. The Km values of LpAAL protein for aspartic acid, fumaric acid, phenylalanine, and tyrosine as substrates were 5.7, 8.5, 4.4, and 1.2 mM, respectively. The kcat values of LpAAL protein for aspartic acid, fumaric acid, phenylalanine, and tyrosine as substrates were 6.7, 0.45, 4.96, and 0.02 s−1, respectively. Therefore, aspartic acid, fumaric acid, phenylalanine, and tyrosine are bona fide substrates for LpAAL enzyme.

Poly-Histidine-Tagged Protein Purification Using Immobilized Metal Affinity Chromatography (IMAC).

His-tagging is the most widespread and versatile strategy used to purify recombinant proteins for biochemical and structural studies. Recombinant DNA methods are first used to engineer the addition of a short tract of poly-histidine tag (His-tag) to the N-terminus or C-terminus of a target protein. The His-tag is then exploited to enable purification of the "tagged" protein by immobilized metal affinity chromatography (IMAC). In this chapter, we describe efficient procedures for the isolation of highly purified His-tagged target proteins from an Escherichia coli host using IMAC in a bind-wash-elute strategy that can be performed under both native and denaturing conditions.

A phase separation process induced by pH change for purification of His-tagged protein at low salt concentration

Protein purification is critical for protein research and enzyme manufacture, and efficient purification methods with low cost have a high priority. In this study, the difference in solubility between His-tagged protein and nontagged protein led to a liquid-solid phase separation at neutral pH and low salt concentration and purified His-tagged protein to some extent. The investigation of impurity in the precipitate indicated that solvation behavior varied from several oligo-amino acid tagged proteins. Among them, proteins with oligo-Lys, oligo-Trp and oligo-Arg obviously precipitated at low salt concentration and weak acidic pH, which is similar to that for the precipitation of His-tagged protein. Further study suggested that proteins with oligo-Lys, oligo-Trp and oligo-Arg remained a precipitate state while His-tagged protein dissolved at a slightly higher pH value. Based on these finding, a method was developed for the purification of His-tagged protein via phase separation induced by pH (PSIP) at low salt concentration. Though the yield and purity of protein purified by PSIP were slightly inferior to that by immobilized metal ion affinity chromatography (IMAC), PSIP was a high-throughput and low-cost method for purification of His-tagged proteins. Beyond the purification process, the various solvation behaviors of proteins with different oligo-amino acid also provide a reference for solvation research of protein.

Functional Characterization of Novel Bony Fish Lipoxygenase Isoforms and Their Possible Involvement in Inflammation.

Eicosanoids and related compounds are pleiotropic lipid mediators, which are biosynthesized in mammals via three distinct metabolic pathways (cyclooxygenase pathway, lipoxygenase pathway, epoxygenase pathway). These mediators have been implicated in the pathogenesis of inflammatory diseases and drugs interfering with eicosanoid signaling are currently available as antiphlogistics. Eicosanoid biosynthesis has well been explored in mammals including men, but much less detailed information is currently available on eicosanoid biosynthesis in other vertebrates including bony fish. There are a few reports in the literature describing the expression of arachidonic acid lipoxygenases (ALOX isoforms) in several bony fish species but except for two zebrafish ALOX-isoforms (zfALOX1 and zfALOX2) bony fish eicosanoid biosynthesizing enzymes have not been characterized. To fill this gap and to explore the possible roles of ALOX15 orthologs in bony fish inflammation we cloned and expressed putative ALOX15 orthologs from three different bony fish species (N. furzeri, P. nyererei, S. formosus) as recombinant N-terminal his-tag fusion proteins and characterized the corresponding enzymes with respect to their catalytic properties (temperature-dependence, activation energy, pH-dependence, substrate affinity and substrate specificity with different polyenoic fatty acids). Furthermore, we identified the chemical structure of the dominant oxygenation products formed by the recombinant enzymes from different free fatty acids and from more complex lipid substrates. Taken together, our data indicate that functional ALOX isoforms occur in bony fish but that their catalytic properties are different from those of mammalian enzymes. The possible roles of these ALOX-isoforms in bony fish inflammation are discussed.

A novel aptamer beacon for rapid screening of recombinant cells and in vivo monitoring of recombinant proteins

Recombinant protein drugs, which are typically produced by mammalian host cells, have been approved for the treatment of a range of diseases. Accordingly, systems for selecting recombinant cell lines with efficient protein expression and for testing the content of recombinant proteins in vivo are crucial to the large-scale production and application of protein-based therapeutic drugs. In this study, we designed three aptamer beacons to detect His-tag, a common label of recombinant proteins. We found that all three beacons could specifically and quantitatively measure the His-tagged recombinant proteins with a short reaction time. Among these three beacons, the 6H5-MU beacon had the highest sensitivity for His polypeptides with a detection limit of 250ng/mL and the shortest detection time within 1min. Furthermore, we established a rapid and highly effective recombinant cell line construction system, which could obtain monoclonal cell lines with high yields of target proteins within 21days, by combining 6H5-MU with pSB, a novel plasmid composed of a Sleeping Beauty transposase and a transposon. Finally, 6H5-MU also discriminately tested the serum concentration of His-tagged recombinant proteins in vivo, with consistent results compared to enzyme-linked immunosorbent assay (ELISA). We thus established a rapid and high-throughput method for generating recombinant cell lines and in vivo monitoring of recombinant protein levels, thereby providing a new platform for the development and preparation of recombinant protein drugs. KEY POINTS: • The 6H5-MU aptamer beacon rapidly and accurately binds to His-tagged recombinant proteins. • A system for rapid and high-throughput generation of recombinant cell lines is established using 6H5-MU and pSB. • 6H5-MU allows in vivo monitoring of recombinant protein levels.

Structural and biochemical insights into His-tag-induced higher-order oligomerization of membrane proteins by cryo-EM and size exclusion chromatography

Structural and functional characterization of proteins as well as the design of targeted drugs heavily rely on recombinant protein expression and purification. The polyhistidine-tag (His-tag) is among the most prominent examples of affinity tags used for the isolation of recombinant proteins from their expression hosts. Short peptide tags are commonly considered not to interfere with the structure of the tagged protein and tag removal is frequently neglected. This study demonstrates the formation of higher-order oligomers based on the example of two His-tagged membrane proteins, the dimeric arginine-agmatine antiporter AdiC and the pentameric light-driven proton pump proteorhodopsin. Size exclusion chromatography revealed the formation of tetrameric AdiC and decameric as well as pentadecameric proteorhodopsin through specific interactions between their His-tags. In addition, single particle cryo-electron microscopy (cryo-EM) allowed structural insights into the three-dimensional arrangement of the higher-order oligomers and the underlying His-tag-mediated interactions. These results reinforce the importance of considering the length and removal of affinity purification tags and illustrate how neglect can lead to potential interference with downstream biophysical or biochemical characterization of the target protein.

Development of alkaline phosphatase-scFv and its use for one-step enzyme-linked immunosorbent assay for His-tagged protein detection.

A histidine (His)-tag is composed of six His residues and typically exerts little influence on the structure and solubility of expressed recombinant fusion proteins. Purification methods for recombinant proteins containing His-tags are relatively well-established, thus His-tags are widely used in protein recombination technology. We established a one-step enzyme-linked immunosorbent assay (ELISA) for His-tagged recombinant proteins. We analyzed variable heavy and light chains of the anti-His-tag monoclonal antibody 4C9 and used BLAST analyses to determine variable zones in light (VL) and heavy chains (VH). VH, VL, and alkaline phosphatase (ALP) regions were connected via a linker sequence and ligated into the pGEX-4T-1 expression vector. Different recombinant proteins with His tags were used to evaluate and detect ALP-scFv activity. Antigen and anti-His-scFv-ALP concentrations for direct ELISA were optimized using the checkerboard method. ZIKV-NS1, CHIKV-E2, SCRV-N, and other His-tag fusion proteins demonstrated specific reactions with anti-His-scFv-ALP, which were accurate and reproducible when the antigen concentration was 50 µg mL-1 and the antibody concentration was 6.25 µg mL-1. For competitive ELISA, we observed a good linear relationship when coating concentrations of recombinant human anti-Müllerian hormone (hAMH) were between 0.78 and 12.5 µg mL-1. Our direct ELISA method is simple, rapid, and accurate. The scFv antibody can be purified using a prokaryotic expression system, which provides uniform product quality and reduces variations between batches.