His-tag Research Articles

HupZ, a Unique Heme-Binding Protein, Enhances Group A Streptococcus Fitness During Mucosal Colonization.

Group A Streptococcus (GAS) is a major pathogen that causes simple and invasive infections. GAS requires iron for metabolic processes and pathogenesis, and heme is its preferred iron source. We previously described the iron-regulated hupZ in GAS, showing that a recombinant HupZ-His6 protein binds and degrades heme. The His6 tag was later implicated in heme iron coordination by HupZ-His6. Hence, we tested several recombinant HupZ proteins, including a tag-free protein, for heme binding and degradation in vitro. We established that HupZ binds heme but without coordinating the heme iron. Heme-HupZ readily accepted exogenous imidazole as its axial heme ligand, prompting degradation. Furthermore, HupZ bound a fragment of heme c (whose iron is coordinated by the cytochrome histidine residue) and exhibited limited degradation. GAS, however, did not grow on a heme c fragment as an iron source. Heterologous HupZ expression in Lactococcus lactis increased heme b iron use. A GAS hupZ mutant showed reduced growth when using hemoglobin as an iron source, increased sensitivity to heme toxicity, and decreased fitness in a murine model for vaginal colonization. Together, the data demonstrate that HupZ contributes to heme metabolism and host survival, likely as a heme chaperone. HupZ is structurally similar to the recently described heme c-degrading enzyme, Pden_1323, suggesting that the GAS HupZ might be divergent to play a new role in heme metabolism.

Towards the First Multiepitope Vaccine Candidate against Neospora caninum in Mouse Model: Immunoinformatic Standpoint.

Neospora caninum is an economically significant parasite among livestock, particularly in dairy cattle herds, causing storm abortions. Vaccination seems necessary to limit the infection and its harsh consequences. This is the first steps towards developing a multiepitope vaccine candidate against N. caninum using in silico approaches. High-ranked mouse MHC-binding and shared linear B-cell epitopes from six proteins (SRS2, MIC3, MIC6, GRA1, IMP-1, and profilin) as well as IFN-γ-inducing epitopes (from SAG1) were predicted, screened, and connected together through appropriate linkers. Finally, RS-09 protein (TLR4 agonist) and histidine tag were added to N- and C-terminal of the vaccine sequence, yielding 486 residues in length. Physicochemical properties showed a stable (instability index: 27.23), highly soluble, antigenic (VaxiJen score: 0.9554), and nonallergenic candidate. Secondary structure of the multiepitope protein included 58.85% random coil, 20.99% extended strand, and 20.16% alpha helix. Also, the tertiary structure was predicted, and further analyses validated a stable interaction between the vaccine model and mouse TLR4 (binding score: -1261.6). Virtual simulation of immune profile demonstrated potently stimulated humoral (IgG+IgM) and cell-mediated (IFN-γ) responses upon multiepitope vaccine injection. Altogether, a potentially immunogenic vaccine candidate was developed using several N. caninum proteins, with the capability to elicit IFN-γ upsurge and other components of cellular immunity, and can be used in prophylactic purposes against neosporosis.

Expression of membrane beta-barrel protein in E. coli at low temperatures: Structure of Yersinia pseudotuberculosis OmpF porin inclusion bodies

The recombinant OmpF porin of Yersinia pseudotuberculosis as a model of transmembrane protein of the β-barrel structural family was used to study low growth temperature effect on the structure of the produced inclusion bodies (IBs). This porin showed a very low expression level in E. coli at a growth temperature below optimal 37 °C. The introduction of a N-terminal hexahistidine tag into the mature porin molecule significantly increased the biosynthesis of the protein at low cultivation temperatures. The recombinant His-tagged porin (rOmpF-His) was expressed in E. coli at 30 and 18 °C as inclusion bodies (IB-30 and IB-18). The properties and structural organization of IBs, as well as the structure of rOmpF-His solubilized from the IBs with urea and SDS, were studied using turbidimetry, electron microscopy, dynamic light scattering, optical spectroscopy, and amyloid-specific dyes. IB-18, in comparison with IB-30, has a higher solubility in denaturants, suggesting a difference between IBs in the conformation of the associated polypeptide chains. The spectroscopic analysis revealed that rOmpF-His IBs have a high content of secondary structure with a tertiary-structure elements, including a native-like conformation, the proportion of which in IB-18 is higher than in IB-30. Solubilization of the porin from IBs is accompanied by a modification of its secondary structure. The studied IBs also contain amyloid-like structures. The results obtained in this study expand our knowledge of the structural organization of IBs formed by proteins of different structural classes and also have a contribution into the new approaches development of producing functionally active recombinant membrane proteins.

Computational design of immunogenic peptide constructs comprising multiple human leukocyte antigen restricted dengue virus envelope epitopes.

Dengue virus (DENV) is endemic in 100 countries with the ability to impact nearly 50% of world population. DENV envelope (E) protein is responsible for viral attachment to host cells and has been target of various countermeasure development efforts. The current study focuses on a consensus computational approach to identify cross-reactive, immunogenic DENV-2 E peptides displaying promiscuity with a wide array of human leukocyte antigen (HLA) molecules. Four conserved peptides (FP-1, FP-2, FP-3 and FP-4) containing multiple CD8+ and CD4+ T cell epitopes were identified by employment of various immunoinformatics tools. FP-1, FP-2, FP-3 and FP-4 were estimated to bind with 227, 1787, 1008 and 834 HLA alleles, respectively. Root mean square deviation (RMSD) values obtained by molecular docking (CABS-Dock) with 20 HLA alleles (10 each of HLA classes I and II) resulted into comparable RMSD values of identified epitopes with native peptides, which represents the natural presentation of epitopes to HLA molecules. These peptides were also found to be part of previous experimentally validated immunogenic peptides. Further, a dengue immunogenic peptide construct was generated by linking the four peptides, an adjuvant and a 6× histidine tag. The construct showed strong binding and stability with Toll-like receptor. Collectively, these results provide strong evidence in the support of the immunogenic potential of the dengue immunogenic peptide construct.

Lysine‐2,3‐aminomutase and a newly discovered glutamate‐2,3‐aminomutase produce β‐amino acids to overcome salt stress in methanogenic archaea

Many methanogenic archaea synthesize N ε ‐acetyl‐β‐lysine and/or β‐glutamate as osmolytes that allow survival in high salinity environments. Methanococcus maripaludis C7 contains two genes, MmarC7_0106 and MmarC7_1783, encoding radical S‐adenosylmethionine(SAM) enzymes with sequence similarity to lysine‐2,3‐aminomutase (KAM). MmarC7_0106 is immediately upstream of an acetyltransferase and was expected to be the KAM involved in the biosynthesis of N ε ‐acetyl‐β‐lysine. To confirm this, the gene was cloned and expressed with a hexahistidine tag in E. coli, followed by anaerobic purification and biochemical characterization. Although the bacterial KAMs involved in lysine degradation in Clostridium subterminale and a post‐translational modification of elongation factor P in E. coli have been well‐characterized, the archaeal KAM involved in compatible solute biosynthesis had never been studied in vitro before. In the presence of L‐lysine, SAM, and dithionite at 37°C, this archaeal KAM had a k cat = 0.42 s ‐1 and a K m = 17.8 mM. The product was shown to be 3(S)‐β‐lysine, which is like the well‐characterized Clostridium KAM as opposed to the E. coli KAM that produces 3(R)‐β‐lysine. The archaeal KAM was highly specific for L‐lysine, with no activity observed with D‐lysine or most other amino acids. However, low aminomutase activity was observed with L‐arginine. We further report the function of MmarC7_1783, a putative radical SAM aminomutase with a ~160 amino acid extension at its N‐terminus. Bioinformatic analysis of possible substrate binding residues suggested a function as glutamate‐2,3‐aminomutase, which was confirmed here through heterologous expression in a methanogen followed by detection of β‐glutamate in cell extracts. Finally, we investigated the differences in amino acid compatible solute usage amongst M. maripaludis C7 (contains both aminomutase genes) and M. maripaludis S2 (contains only the canonical KAM gene) in different salt concentrations. Taken together, this work defines the biosynthetic routes for β‐amino acids in methanogenic archaea and expands the importance and diversity of radical SAM enzymes in all domains of life.

Expression and characterization of a putative cyclooxygenase from the genome of Nitrosospira multiformis

Cyclooxygenases are dual‐function heme proteins critical for the biosynthesis of prostaglandins in mammals. Mammalian cyclooxygenases contain both dioxygenase activity (adding molecular oxygen to lipid substrates to form lipid hydroperoxides) and peroxidase activity (reducing the hydroperoxides to alcohols). Two open reading frames in the ammonia‐oxidizing betaproteobacterium Nitrosospira multiformis are annotated as cyclooxygenases by structural prediction of the protein product. We have attempted to express and characterize one of these open reading frames to test the structural prediction. DNA encoding the desired open reading frame was synthesized and inserted into several expression vectors with N‐terminal fusion partners (his6 tag, SUMO, thioredoxin). Protein expression yielded largely insoluble protein product under many different expression conditions. Recently, we have been able to express a thioredoxin‐Nitrosospira cyclooxygenase fusion partner in soluble form. Ongoing experiments to characterize the protein structurally and enzymatically will be reported.

Tagging Polyphosphate Kinase Alters Enzymatic Activity and Affect Stress Recovery in E. coli

Many bacterial species, including Escherichia coli (E. coli) utilize the enzyme polyphosphate kinase (PPK) to synthesize polyphosphate (polyP) in response to biological stress. Multiple studies have shown that impairing PPK activity impairs bacterial pathogenicity and survival, which has made it a target for therapeutic development. Unfortunately, PPK regulation is poorly understood. To address this, we previously described a series of mutations in E. coli ppk, termed ppk*, that result in high polyP accumulation in vivo. However, the specific activity of PPK* enzyme in vitro, when purified using a C‐term 6X His‐tag (PPK*‐HT), was comparable to purified WT enzyme (PPK‐HT).PPK activity is tied to its oligomeric state, with the dimeric form synthesizing polyP. To determine if the ppk* mutation altered oligomerization, we tested PPK‐HT and PPK*‐HT enzymes in an analytical ultracentrifuge. We found that the PPK‐HT enzyme was mostly dimeric but had a small monomeric population. Conversely, the PPK*‐HT enzyme was exclusively dimeric. This suggested that the ppk* mutation may affect oligomerization, but the His‐tag masked any effect on activity.We next hypothesized that the C‐term His tag alters the in vitro activity. To test this, we compared the specific activities of PPK‐HT and PPK*‐HT to that of PPK purified using a smaller C‐term C tag (PPK‐CT) and PPK enzyme purified with no tags (PPK‐NT). PPK*‐HT and PPK‐HT had comparable activities in vitro but were significantly more active than the untagged enzyme. PPK‐CT and PPK‐NT had comparable activities. This suggests that the C‐tag does not alter PPK activity while the His‐tag does.We next tested the purified enzymes’ sensitivity to substrate by testing PPK activity in 6mM and 20mM ATP. We found that the His‐tag increased the enzyme’s activity in the higher ATP concentrations. Comparing the 20mM and 6mM samples, the His‐tag enzymes showed an increase in activity of ~3.5 fold compared to their 6mM conditions. Conversely, the PPK‐CT and PPK‐UT only showed a 1.4‐ and 1.9‐ fold increase in activity, respectively.PolyP synthesis impairs bacterial growth and replication. We hypothesized that a more active PPK would impair recovery following a prolonged stationary phase. We developed strains carrying one of four expression vectors: an empty vector control (EV), PPK‐HT, PPK‐CT, or native PPK. Using these four strains, we developed a bacterial growth recovery curve assay. We found that while the PPK‐CT and untagged PPK strains had comparable recovery and growth rates, the PPK‐HT strain had a slower recovery rate resulting in a later shift into log phase.Taken together, our results have significant implications for polyP biology. We have shown that the standard purification method produces an enzyme with altered substrate sensitivities, making it a poorer candidate for drug development studies. C‐tag enzyme is comparable to untagged enzyme, making it a reliable candidate for future studies. Finally, the in vitro differences produce an in vivo phenotype, making it easy to test the effect of the tags in living systems.

Comparison of Prostate‐Specific Transglutaminase Function in the Great Apes

The copulatory plug is a hardened structure formed through semen coagulation post‐ejaculation which is theorized to protect against remating, stimulate the female reproductive tract, and aid in sperm release and transport. This structure is prevalent in species that experience high levels of sperm competition including chimpanzees and several other primates. Chimpanzees experience high levels of sperm competition due to their polygynandrous mating system. In polygynandry females will mate with multiple partners during an estrus cycle leading to direct competition between the sperm of each male to fertilize the same egg. This varies distinctly from polygynous and monogamous species, such as humans, where females mate with only one male and sperm competition is diminished. Based on previous work in rats, prostate‐specific transglutaminase 4 (TGM4) is implicated as the driving force of the formation of the copulatory plug. TGM4 is a member of the transglutaminase family of enzymes which catalyze the calcium‐dependent formation of an isopeptide bond between glutamine and lysine residues. While humans do not experience a great deal of sperm competition or form a copulatory plug, human semen goes through a similar process of post‐ejaculatory coagulation which is predicted to be carried out by TGM4 under the same mechanism. Interestingly, there are only seven amino acid differences between the human and chimpanzee TGM4s and yet the degree of coagulation between the two species is vastly different. The aim of this study to characterize and compare the function of these proteins to determine the major contributing factor in semen coagulation. The primary hypothesis was that chimpanzee TGM4 would have higher enzymatic activity and more readily cross‐link artificial substrates than human TGM4. To test this hypothesis, the coding sequences were acquired for human and chimpanzee TGM4 as well as eight other primate species. Using a maximum likelihood‐based method, we generated a gene tree of primate TGM4 and calculated dN/dS ratios for each branch. Human TGM4 had a dN/dS ratio of 1.04 suggesting neutral evolution while chimpanzee TGM4 had a ratio of >1 suggesting strong positive selection. We further predicted the sequence of TGM4 of the last human‐chimp ancestor using ancestral sequence reconstruction which utilizes the known phylogenetic tree and a maximum likelihood approach with the software package Phylogenetic Analysis by Maximum Likelihood (PAML). Coding sequences representing TGM4 from humans, chimpanzees, and the last human‐chimp ancestor were cloned into a mammalian expression vector containing a C‐terminal His6tag and were transfected into HEK‐293T cells. Purified TGM4 was used in an in‐vitro colorimetric enzymatic assay to quantify enzymatic activity. Our results suggest that mating system variation has led to differences in both sequence and function of seminal proteins. These functional differences speak to the impact of even a small number of mutations on protein function as well as the influence of environmental pressures on protein evolution.

Sperm Competition and Its Effects on the Evolution of Prostatic Acid Phosphatase in Hominids

Selective pressures and their effects on protein structure and function can be found by comparing genes or proteins from different organisms. These pressures can take varying forms. Sexual selection, a form of natural selection, occurs when an organism acquires traits that give it an advantage in mating and producing offspring. An example can be seen in hominid seminal proteins. Hominids exhibit different mating systems which has resulted in seminal proteins being affected by varying degrees of sperm competition. This can result in variation both in the sequences of these genes and the function of the gene products. One such gene, ACPP, is used to generate the protein prostatic acid phosphatase (PAP). This protein is known to dephosphorylate a number of targets in seminal plasma, and it functions as a phosphatase in the acidic pH range. It has been suggested computationally by comparing dN/dS ratios that ACPP may be under strong positive selection in chimpanzees (a species with high sperm competition) and purifying selection in humans and gorillas (both species with weaker sperm competition). We hypothesize that varying degrees of sperm competition have resulted in sequence and functional variation at ACPP/PAP among hominids. ACPPcoding sequences for human, chimp, and gorilla homologs were each inserted into a mammalian expression vector with a C‐terminal His6 tag and transfected into HEK‐293T cells. The phosphatase activities of the PAP homologs were compared using a DiFMUP fluorescent substrate assay. Each protein was tested for pH optima, phosphatase activity at a fixed substrate concentration, and kcat/km. The PAP orthologs exhibited similar pH profiles, with a pH optimum between pH 5.0‐6.0. However, it was found that chimp PAP exhibited elevated phosphatase activity when compared to the other hominids, and this was consistent across a varying pH range. These results suggest that positive selection of chimp ACPPhas resulted in a more efficient PAP protein with elevated activity when compared to human and gorilla PAP, perhaps due to strong sperm competition. Gorilla PAP showed the lowest activity, which is interesting considering that many genes that code for male reproductive proteins have become pseudogenes in gorillas, a species with very low sperm competition. Overall, the results suggest that the varying mating systems of the hominid primates have resulted in functional differences in the PAP homologs of humans, chimpanzees, and gorillas. By bridging together functional and computational techniques such as in this research, we are beginning to understand the effects of specific selective pressures on the function of male reproductive proteins.

Mass-Encoded Suspension Array for Multiplex Detection of Matrix Metalloproteinase Activities.

This work designed a mass spectrometric biosensing strategy for the multiplex detection of matrix metalloproteinases (MMPs) with a mass-encoded suspension array. This array was fabricated as multiplex sensing probes by functionalizing magnetic beads with MMP-specific peptide-isobaric tags for relative and absolute quantification (iTRAQ) conjugates, which contained a hexahistidine tag for surface binding, a substrate region for MMP cleavage, and a coding region for the specific MMP. The integration of the multiplex coding ability of iTRAQ with ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and the proteolysis method for peptide digestion endowed the biosensing method with high throughput and ultrahigh sensitivity. This strategy could be conveniently performed by mixing the sample and the suspension array for enzymatic reactions and then digesting the uncleaved peptides with trypsin to release the coding regions for UPLC-MS/MS analysis. With MMP-2 and MMP-7 as analytes, the relative changes of peak area ratios of coding regions showed good linear responses in the ranges of 0.2-100 and 0.5-400 ng mL-1, with detection limits of 0.064 and 0.17 ng mL-1, respectively. The analysis of MMP activity in serum samples and its change responding to inhibitors demonstrated the specificity, practicability, and expansibility of the proposed strategy. This work paves a new avenue for the activity assays of multiplex enzymes and promotes the development of mass spectrometric biosensing.

Expression and Purification cry1Aa Gene

The cry gene encodes a crystal protein of Bacillus thuringiensis known as Cry toxin, which is toxic to major Lepidopteran insect pest. The effectiveness of Cry toxin against the rice yellow stem borer (YSB, Scirpophaga incertulas Wlk) has been reported. Transgenic Bacillus thuringiensis (Bt) rice has been developed by introducing the fusion gene cry1B::cry1Aa gene driven by the CaMV35S promoter to Javanica rice cv. Rojolele to improve its resistance against rice YSB. Resistance to YSB at a greenhouse and multi-site scales have been conducted and reported. Food and feed safety studies for the release and commercialization of genetically modified crops require a sufficient amount of purified protein sample. Therefore, a strategy to produce Cry1Aa protein in a short time with high productivity needs to be developed. In this study, the cry1Aa gene (1857 bp) was cloned into pRHA-SDM expression vector under the control of the pRHA promoter fused to a 6xHis tag on the C-terminal to produce pRHA-SDM-cry1Aa. The expression host used was Escherichia coli strain NiCo21, and protein purification was performed using IMAC Co2+ on the TALON® matrix. The results showed that the recombinant Cry1Aa protein, approximately 69 kDa, was detected using Western blot performed using anti-rabbit Cry1Aa polyclonal antibody and His detector nickel-NTA conjugated with HRP reporter enzyme. Expression and purification protocols that have been developed can be used to produce Cry1Aa protein which can be utilized for further protein studies.

Soluble Diphtheria Toxin Variant, CRM 197 was Obtained in Escherichia coli at High Productivity Using SUMO Fusion and an Adjusted Expression Strategy.

CRM197, a non-toxic diphtheria toxin variant, is widely used as a polysaccharide carrier in a variety of conjugate vaccines and also exhibits antitumor activity. CRM197 commercial production is limited due to the low yield of Corynebacterium diphtheriae C7 (197) tox-. Developing an efficient method for recombinant CRM197 production reduces production costs and is critical for expanding the application coverage of related medical products and basic research. Escherichia coli is a frequently used host for heterologous protein synthesis. However, the primary limitation of this system is the inclusion body formation and the low yield of active protein recovery. As a result, we attempted to produce CRM197 in the soluble form in E. coli using a small ubiquitin-related modifier (SUMO) tag fusion and an expression strategy optimized for protein production. CRM197 was expressed intracellularly in E. coli BL21 (DE3) with its N-terminus fused to a SUMO tag preceded by a histidine tag (HSCRM197). To improve the solubility of HSCRM197 in E. coli, a response surface method (RSM) experimental design was used based on three factors: expression temperature, inducer concentration, and sorbitol inclusion in the culture medium. Metal affinity chromatography was used to purify HSCRM197, and the SUMO tag was removed using the SUMO protease's catalytic domain. After adsorbing the SUMO tag on a Ni-NTA column, CRM197 was obtained. DNA degradation activity was determined for both HSCRM197 and CRM197. When HSCRM197 was expressed in E. coli under common expression conditions (37ºC, 1000 μM inducer), 15.4% of the protein was found in the cellular soluble fraction. However, when the RSM-derived expression conditions were used (30ºC, 510 μM inducer, and 200 mM sorbitol), the obtained HSCRM197 was almost completely soluble (96.5% solubility), and the system productivity was 32.67 μg ml-1 h-1. HSCRM197 and CRM197 both exhibited nuclease activity. However, the activity of CRM197 was greater than that of HSCRM197. These findings established the utility of the method developed in this study to produce CRM197 for medical applications.

Expression of influenza B virus hemagglutinin and its immunogenicity determination

Influenza B virus is one of the causes for seasonal influenza, which can account for serious illness or even death in some cases. We tested the expression of extracellular domain of hemagglutinin (HA-ecto) of influenza B viruses in mammalian cells, and then determined the immunogenicity of HA-ecto in mice. The gene sequence encoding influenza B virus HA-ecto, foldon sequence, and HIS tag was optimized and inserted into pCAGGS vector. The opening reading frame (ORF) of neuraminidase was also cloned into pCAGGS. The pCAGGS-HA-ecto and pCAGGS-NA were co-transfected into 293T cells using linear polyethylenimine. Cell supernatant after transfection was collected after 96 h, and the secreted trimmeric HA-ecto protein was purified by nickel ion affinity chromatography and size exclusion chromatography. Subsequently, the mice were immunized with HA-ecto protein, and the corresponding antibody titers were detected by ELISA and hemagglutination inhibition (HAI) assays. The results showed that soluble trimeric HA-ecto protein could be obtained using mammalian cell expression system. Moreover, trimeric HA-ecto protein, in combination with the adjuvant, induced high levels of ELISA and HAI antibodies against homogenous and heterologous antigens in mice. Thus, the soluble HA-ecto protein expressed in mammalian cells could be used as a recombinant subunit vaccine candidate for influenza B virus.

Expression and purification of DYRK1A kinase domain in complex with its folding intermediate-selective inhibitor FINDY

The kinase DYRK1A phosphorylates substrate proteins that are involved in the progression of many diseases. DYRK1A also phosphorylates its own residues on key elements intramolecularly to activate and stabilize itself during the folding process. Once the folding process of DYRK1A has completed, it can no longer catalyzes the intramolecular reaction, suggesting that a transitional intermediate state that catalyzes the autophosphorylation exists. In the previous study, we identified a small molecule, designated as FINDY, that selectively inhibits the folding intermediate of DYRK1A. Although evidence has suggested that FINDY targets the ATP-binding pocket of DYRK1A, it remains elusive as to whether the DYRK1A kinase domain could be purified as a complex with FINDY. In this study, we successfully expressed and purified the kinase domain of DYRK1A in complex with FINDY. The DYRK1A kinase domain was expressed as a fusion protein with a hexahistidine tag and ZZ-domain (His-ZZ-DYRK1A) at 6 °C by using a cold shock induction system in Escherichia coli cells. The cells were incubated with FINDY. The cell pellets were gently extracted on ice and subjected to immobilized-metal affinity chromatography. The amount of FINDY in the elution fraction was measured by UV absorbance specific for FINDY. The eluate contained FINDY with the ratio of FINDY to DYRK1A protein being 0.15 in quadruplicate experiments. Thus, this study demonstrates the direct interaction between the DYRK1A kinase domain and FINDY, paving the way for structural determination of the complex.

Transition Metals Induce Quenching of Monomeric Near-Infrared Fluorescent Proteins.

Transition metals such as zinc and copper are essential in numerous life processes, and both deficiency and toxic overload of these metals are associated with various diseases. Fluorescent metal sensors are powerful tools for studying the roles of metal ions in the physiology and pathology of biological systems. Green fluorescent protein (GFP) and its derivatives are highly utilized for protein-based sensor design, but application to anaerobic systems is limited because these proteins require oxygen to become fluorescent. Bacteriophytochrome-based monomeric near-infrared fluorescent proteins (miRFPs) covalently bind a bilin cofactor, which can be added exogenously for anaerobic cells. miRFPs can also have emission wavelengths extending to >700 nm, which is valuable for imaging applications. Here, we evaluated the suitability of miRFP670 and miRFP709 as platforms for single fluorescent protein metal ion sensors. We found that divalent metal ions like Zn2+, Co2+, Ni2+, and Cu2+ can quench from ∼6-20% (Zn2+, Co2+, and Ni2+) and up to nearly 90% (Cu2+) of the fluorescence intensity of pure miRFPs and have similar impacts in live Escherichia coli cells expressing miRFPs. The presence of a 6× histidine tag for purification influences metal quenching, but significant Cu2+-induced quenching and a picomolar binding affinity are retained in the absence of the His6 tag in both cuvettes and live bacterial cells. By comparing the Cu2+ and Cu+-induced quenching results for miRFP670 and miRFP709 and through examining absorption spectra and previously reported crystal structures, we propose a surface metal binding site near the biliverdin IXα chromophore.

The Poly-Histidine Tag H6 Mediates Structural and Functional Properties of Disintegrating, Protein-Releasing Inclusion Bodies.

The coordination between histidine-rich peptides and divalent cations supports the formation of nano- and micro-scale protein biomaterials, including toxic and non-toxic functional amyloids, which can be adapted as drug delivery systems. Among them, inclusion bodies (IBs) formed in recombinant bacteria have shown promise as protein depots for time-sustained protein release. We have demonstrated here that the hexahistidine (H6) tag, fused to recombinant proteins, impacts both on the formation of bacterial IBs and on the conformation of the IB-forming protein, which shows a higher content of cross-beta intermolecular interactions in H6-tagged versions. Additionally, the addition of EDTA during the spontaneous disintegration of isolated IBs largely affects the protein leakage rate, again protein release being stimulated in His-tagged materials. This event depends on the number of His residues but irrespective of the location of the tag in the protein, as it occurs in either C-tagged or N-tagged proteins. The architectonic role of H6 in the formation of bacterial IBs, probably through coordination with divalent cations, offers an easy approach to manipulate protein leakage and to tailor the applicability of this material as a secretory amyloidal depot in different biomedical interfaces. In addition, the findings also offer a model to finely investigate, in a simple set-up, the mechanics of protein release from functional secretory amyloids.

Cloning and Purification of L-Asparaginase from Enterobacter carcerogenus

In this study, the gene coding for EcL-ASP from Enterobacter carcerogenus was identified in full sequence and cloned into a mesophilic organism. The gene encoding L-asparaginase was transferred to the pET-28a (+) vector to ensure its expression in Escherichia coli BL21 (DE3) pLysS. The recombinant protein containing the N-terminal histidine tail (6xHis tag) was purified by Nickel affinity chromatography. As a result of SDS-PAGE, it was determined that the purified protein consisted of a single type of polypeptide. In the theoretical calculation, the subunit molecular weight of the recombinant protein containing the histidine tail was found to be 37 kDa. It was observed that the cloned enzyme had low L-glutaminase activity. The pH and temperature at which the recombinant enzyme showed the best activity were determined as 7.0 and 37 °C, respectively. From the drawn Lineweaver-Burk graph, it is estimated that the Km value is 0.06 mM and the Vmax value is 666.7 U mg-1 protein.

Development and evaluation of indirect enzyme-linked immunosorbent assay using recombinant dense granule antigen 7 protein for the detection of Toxoplasma gondii infection in cats in Thailand.

Background and Aim:Toxoplasma gondii is recognized as a zoonosis causing toxoplasmosis in animals globally. Cat is a definitive host of T. gondii and sheds oocyst through feces, which can infect human beings and animals through contaminated food ingestion. A precise diagnostic test is essential to prevent T. gondii infection in both humans and animals. This study aimed to develop and evaluate the pETite-dense granule antigen 7(GRA7)-based indirect enzyme-linked immunosorbent assay (ELISA) to detect T. gondii infection in cats.Materials and Methods:T. gondii-GRA7 was cloned and expressed in the Expresso®small ubiquitin-related modifier (SUMO) T7 Cloning and Expression System. The recombinant pETite-GRA7 was purified using HisTrap affinity chromatography and confirmed using Western blot analysis. The recombinant protein was used to develop and evaluate the indirect ELISA for T. gondii infection detection. In total, 200 cat sera were tested using pETite-GRA7-based indirect ELISA and indirect fluorescent antibody test (IFAT). The statistical analysis based on Kappa value, sensitivity, specificity, positive predictive value, negative predictive value, χ2 test, and receiver operating characteristic (ROC) curve was used to evaluate the performance of the test.Results:A 606 bp GRA7 polymerase chain reaction (PCR) product was obtained from T. gondii RH strain genomic DNA. The gene was cloned into the pETite™ vector and transformed to HI-Control Escherichia coli BL21 (DE3) for protein expression. Approximately 35 kDa of recombinant pETite-GRA7 was observed and Western blot analysis showed positive bands against anti-6-His antibody and positive-T. gondii cat serum. A sample of 0.5 μg/mL of pETite-GRA7 was subjected to indirect ELISA to detect T. gondii infection in the cat sera. The results showed sensitivity and specificity of pETite-GRA7-based indirect ELISA at 72% and 96%, respectively. An acceptable diagnostic performance was characterized by high concordant results (94%) and substantial agreement (Kappa value=0.65) with IFAT. The seroprevalence levels of ELISA and IFAT were 10% and 9%, respectively, and were not significantly (p>0.05) different. The expected performance of ELISA at different cutoff points using the ROC curve analysis revealed 89% sensitivity and 92% specificity at the cutoff value of 0.146, with a high overall assay accuracy (area under the curve=0.94).Conclusion:In this study, the pETite™ vector, N-terminal 6xHis SUMO fusion tag, was used to improve the solubility and expression level of GRA7. The recombinant pETite-GRA7 showed enhanced protein solubility and purification without special condition requirements. This pETite-GRA7-based indirect ELISA showed high concordant results and substantial agreement with IFAT. ELISA revealed an acceptable sensitivity and specificity. These initial data obtained from cats’ sera demonstrated that pETite-GRA7-based indirect ELISA could be a useful method for local serological diagnosis of T. gondii infection in cats in Thailand.

Metal-Binding Propensity in the Mitochondrial Dynamin-Related Protein 1.

Dynamin-related protein1 (Drp1) functions to divide mitochondria and peroxisomes by binding specific adaptor proteins and lipids, both of which are integral to the limiting organellar membrane. In efforts to understand how such multivalent interactions regulate Drp1 functions, in vitro reconstitution schemes rely on recruiting soluble portions of the adaptors appended with genetically encoded polyhistidine tags onto membranes containing Ni2+-bound chelator lipids. These strategies are facile and circumvent the challenge in working with membrane proteins but assume that binding is specific to proteins carrying the polyhistidine tag. Here, we find using chelator lipids and chelator beads that both native and recombinant Drp1 directly bind Ni2+ ions. Metal binding, therefore, represents a potential strategy to deplete or purify Drp1 from native tissue lysates. Importantly, high concentrations of the metal in solution inhibit GTP hydrolysis and renders Drp1 inactive in membrane fission. Together, our results emphasize a metal-binding propensity, which could significantly impact Drp1 functions.

Engineering Polyhistidine Tags on Surface Proteins of Acidithiobacillus ferrooxidans: Impact of Localization on the Binding and Recovery of Divalent Metal Cations.

Metal processing using microorganisms has many advantages including the potential for reduced environmental impacts as compared to conventional technologies.Acidithiobacillus ferrooxidansis an iron- and sulfur-oxidizing chemolithoautotroph that is known to participate in metal bioleaching, and its metabolic capabilities have been exploited for industrial-scale copper and gold biomining. In addition to bioleaching, microorganisms could also be engineered for selective metal binding, enabling new opportunities for metal bioseparation and recovery. Here, we explored the ability of polyhistidine (polyHis) tags appended to two recombinantly expressed endogenous proteins to enhance the metal binding capacity of A. ferrooxidans. The genetically engineered cells achieved enhanced cobalt and copper binding capacities, and the Langmuir isotherm captures their interaction behavior with these divalent metals. Additionally, the cellular localization of the recombinant proteins correlated with kinetic modeling of the binding interactions, where the outer membrane-associated polyHis-tagged licanantase peptide bound the metals faster than the periplasmically expressed polyHis-tagged rusticyanin protein. The selectivity of the polyHis sequences for cobalt over copper from mixed metal solutions suggests potential utility in practical applications, and further engineering could be used to create metal-selective bioleaching microorganisms.