His-tag Research Articles

Programing Green Light-Responsive Hydrogels: from Photo-Weakening to Photo-unresponsiveness and Photo-Strengthening.

Light-responsive hydrogels are highly valued for their dynamic mechanical properties and biocompatibility. In this study, we present a hydrogel system that can either soften or strengthen on green light exposure, or remain unresponsive to light, depending on the addition of adenosyl cobalamin (AdoCbl) and Co2+. These protein-based hydrogels were formed using genetically encoded SpyTag-SpyCatcher chemistry and included green light-sensitive CarHc protein domains. As previously reported, these hydrogels formed in the dark with the addition of AdoCbl, due to the tetramerization of the CarHc domains. Under green light exposure, the CarHc tetramers disassembled, leading to a rapid transition from gel to sol. Interestingly, we discovered that an excess of AdoCbl leads to photo-strengthening rather than photo-weakening. This occurred because light exposure induces interchain crosslinks between AdoCbl and poly-histidine tags (His6-tags) of the proteins. Furthermore, incorporating Co2+ ions enhanced hydrogel stiffness by coordinating to His6-tags. This not only suppressed photo-weakening but also promoted photo-strengthening behaviour. These findings highlight the role of His6-tags in photochemical crosslinking with excess AdoCbl and in coordination to Co2+ ions, providing a novel strategy for designing tuneable, light responsiveness in materials.

A Structural Bioinformatics-Guided Study of Adenosine Triphosphate-Binding Cassette (ABC) Transporters and Their Substrates

Adenosine triphosphate-binding cassette (ABC) transporters form a ubiquitous superfamily of integral membrane proteins involved in the translocation of substrates across membranes. Human ABC transporters are closely linked to the pathogenesis of diseases such as cancer, metabolic diseases, and Alzheimer’s disease. In this study, four ABC transporters were chosen based on (I) their importance in humans and (II) their score in a structural bioinformatics screen aimed at the prediction of crystallisation propensity. The top-scoring ABC transporters’ orthologs (Mus musculus—mouse ABCB5, Ailuropoda melanoleuca—giant panda ABCB6, Myotis lucifugus—little brown bat ABCG1 and Mus musculus ABCG4) were then expressed in Saccharomyces cerevisiae with a combined green fluorescent protein and polyhistidine tag, enabling visualisation and purification. After partial purification and in the presence of the detergent (n-dodecyl-β-D-maltoside), the kinetic parameters of the ATP hydrolysis reactions of the orthologs were determined, as well as the extent of stimulation of their activity when presented with putative substrates. We discuss the efficiency of such bioinformatics approaches and make suggestions for their improvement and wider application in membrane protein-structure determination.

Recombinant Protein Production and Purification of Rift Valley Fever Virus Nucleoprotein from Escherichia coli Expression Systems.

The recombinant expression and purification of viral proteins are a key component in the study of the immune response of viruses, as well as the creation of diagnostic techniques for the detection of viruses. For structurally simple proteins, one commonly used technique is the production of recombinant proteins in bacterial expression systems, which enable the large-scale synthesis and purification of recombinant viral proteins. In this technique, the cDNA encoding for a viral protein is cloned into a bacterial expression vector (with an appropriate purification tag), produced in a modified bacterial culture, and optimized for maximum protein production in a minimal amount of time. In this chapter, a protocol for the production of Rift Valley fever virus nucleoprotein is described. This protein has been previously shown to highly antigenic (and thus, is used in diagnostic tests), and has also been shown to be a potent inducer of the T-cell response following Rift Valley fever virus infection. The protocol outlined in this chapter describes the cloning of the cDNA of RVFV nucleoprotein into a bacterial expression vector, which also contains a fusion protein for optimal protein expression and solubilization, as well as a poly-histidine tag for efficient purification This chapter also describes the steps required for bacterial transformation, culture, lysis, purification and dialysis of RVFV nucleoprotein, resulting in a recombinant protein preparation, which can be upscaled to produce milligram quantities of protein product, which in turn can be used for downstream immunological and diagnostic applications.

Development of high-performance inducible and secretory expression vector and host system for enhanced recombinant protein production

The production of lipopolysaccharide (LPS)-free recombinant proteins from culture supernatants is of great interest to biomedical research and industry. Due to the LPS-free cell wall structure and the well-defined secretion factor B (SecB)-dependent secretion pathway, Gram-positive bacteria are a superior alternative to Escherichia coli expression systems. However, the lack of inducible expression systems for high yields has been a bottleneck. To address this, we developed the pKS81 plasmid, featuring the uhpT (glucose-6-phosphate [G6P] transporter) promoter for high expression of recombinant proteins induced by extracellular G6P via a three-component hexose phosphate transport regulatory system (HptARS), the N-terminal SecB-dependent signal peptide sequence for recombinant protein secretion, and the C-terminal 8 × histidine tag for purification by nickel affinity chromatography. We also generated an expression host strain, Staphylococcus aureus LAC9, lacks the uhpT gene and harmful superantigen and leukotoxin genes, allowing for constitutive HptARS activation by extracellular G6P and increased safety, respectively. Using the pKS81 plasmid, we successfully achieved high yields of prokaryotic (staphylococcal leukotoxin E) and eukaryotic (human annexin A2 protein tagged with mouse IgG1) recombinant proteins, up to 900 mg/L. Our newly established inducible and secretory expression system provides for efficient production and easy purification of LPS-free recombinant proteins, making it valuable for biomedical research and industrial applications.

Designing a Multi-Epitope Candidate Vaccine against Zika Virus NS5 Protein Through Immunoinformatics Approach for Producing in Plant Systems

Background: The Zika virus is an infectious virus that belongs to the Flaviviridae family. It is transmitted to humans through mosquito vectors and poses a serious threat to human health. Despite available resources, there is currently no effective and secure vaccine against the Zika virus to prevent such infections Objective: Observation of symptoms including fever, rash, joint pain, and conjunctivitis (red eyes) along with the possibility of severe complications during pregnancy, including birth defects such as microcephaly, is noted in infected individuals. So, it is the need of the hour to develop a potential vaccine that can immunize the human beings against this virus and protect them. For the purpose of vaccine development against the Zika virus, bioinformatics and immunoinformatics approaches are utilized. Materials and methods: Different epitopes from RdRp Protein of Zika virus were predicted including B-Cell and T- Cell epitopes. The selected epitopes were analyzed further in order to check their antigenicity, immunogenicity, solubility and toxicity in correspondence to their ability to initiate immune response in the individuals. The potential epitopes having the ability to trigger a good immune response and having non- toxic nature were utilized for vaccine development. By combining the different epitopes, a potential multi-epitope vaccine construct was developed. Combining of epitopes was done using EAAAK, AAY and GPGPG linkers. A 50S ribosomal protein was added at N terminal to improve the immunogenicity of the vaccine and 6-His tag protein also added at C terminal. Results: The constructed vaccine was found to have good population coverage all across the world. In molecular docking, strong interactions were found between the constructed vaccine and Toll-like receptor 3 (-346.16), suggesting their potential relevance in the immune response to the vaccine and dynamical stabilities were inspected through molecular dynamic (MD) simulation methods. The developed vaccine construct also exhibited good results of immune response in immune simulation and revealed that the constructed vaccine activates B and T lymphocytes which induce high levels of antibodies and cytokines to combat Zika infection. The constructed vaccine is an effective biomarker with non-toxicity, non-allergic properties, good immunogenicity, and antigenicity. However, experimental assays are required to verify the results of the present study. Conclusion: These findings suggest that design vaccine candidate effectively trigger immune response with no side effects against Zika virus. Keywords:- Zika virus vaccine, Epitope prediction, immunoinformatics, Multiepitope vaccine, Molecular docking.

Effect of C- and N-Terminal Polyhistidine Tags on Aggregation of Influenza A Virus Nuclear Export Protein

Effect of C- and N-Terminal Polyhistidine Tags on Aggregation of Influenza A Virus Nuclear Export Protein

Development of an In Vivo, Real-Time and Continuous Insulin Sensor Utilizing an Extended Gate Field Effect Transistor-Based Transducer

Introduction In this paper, we aim to utilize an extended gate field-effect transistor (EGFET)-based transducer to measure insulin. The design of EGFETs is characterized by a unique structural arrangement, where a physical barrier exists between the semiconductor component and the analyte or physical medium. Due to this structure, EGFET possesses the advantage of protecting the electrode from intricacies within the measurement solution or medium. The need for a redox probe is also effectively eliminated by incorporating EGFETs, marking a significant breakthrough in electrochemical measurement. The lack of a redox probe allows EGFET-based sensors to conduct measurements with minimal invasiveness and enhanced biocompatibility in accordance with an in vivo monitoring system. As the target analyte is not required to be electrochemically active, EGFET is highly customizable for a wide range of targets, including neurotransmitters, biomarkers, and metabolites. Therefore, we constructed immunosensors immobilized with anti-insulin single-chain antibody (scFv) and measured insulin levels, with the objective of creating an in vivo sensor designed to improve glycemic management. We aimed to implement the inherent semiconductor properties of EGFETs by performing real-time monitoring of changes in electrical properties to quantify various insulin concentrations. Methods Development of an in vivo sensor requires the use of a platform that can be implemented within an in vivo environment. For this purpose, we utilized a gold microwire electrode immobilized with anti-insulin scFv. The gold microwire electrodes were prepared by electrochemically cleaning and further roughening them to increase the active surface area for the purpose of improving immobilization of the antibody. To achieve site-specific immobilization of anti-insulin scFv, we employed a nickel-chelated nitrilotriacetate self-assembled monolayer (Ni-NTA SAM). As the scFv harbors a histidine tag and Ni-NTA has an affinity for histidine residues, we were able to achieve oriented immobilization on the electrode surface. Within this study, we compared the effects of covalent immobilization, which leads to random orientation with the antibody, and non-covalent oriented immobilization by comparing the signal obtained from EGFET measurement. EGFET measurements were conducted using a transistor consisting of source, drain, and gate terminals. In this configuration, a constant potential was maintained between the source and drain terminals, while a sweeping potential was applied between an Ag/AgCl reference electrode and the source terminal. Measurement of insulin was performed within a buffer and artificial interstitial fluid (aISF) to model an in vivo environment. Results and Discussion The interaction between insulin and the immobilized anti-insulin scFv on the electrode surface induces a shift in surface potential, driven by the redistribution of charges across the extended gate. The change in surface potential leads to a subsequent modification of the gate potential, known as Vg. The gate potential, when less than the threshold voltage (Vth), operates in the cutoff region where little to no current is present. However, when Vg is greater than Vth, the system operates in the ohmic region where current can flow. The following relationship allowed us to monitor the drain-source current or Ids within the ohmic region to evaluate insulin concentrations. The signal change due to insulin binding to anti-insulin scFv was observed at a reference voltage of 0.6 volts. Conclusion We measured insulin concentrations using an electrode modified with anti-insulin scFv and demonstrated that EGFET can be used as a working principle for an in vivo monitoring system. Additionally, the measurement principle does not require a redox probe within the measurement solution. Future work will include implanting the anti-insulin scFv immobilized gold microwire electrode within a rat model and measuring insulin.

Recombinant human FOXJ1 protein binds DNA, forms higher-order oligomers, has gel-shifting domains and contains intrinsically disordered regions

Forkhead box protein J1 (FOXJ1) is the key transcriptional regulator during the conversion of mammalian primary cilium with a 9 + 0 architecture to the motile (9 + 2) one. The nucleotide sequences of the full-length and DNA-binding domain (DBD) of the open reading frame (ORF) were isolated and expressed into E. coli as 6xHis-tagged proteins. Upon induction, the DBD formed inclusion bodies that solubilized with 8 M urea. No induction of 6xHis-FOXJ1 protein was seen despite sub-cloning into several expression vectors and E. coli host strains. To improve induction and solubility, the 6xHis tag was substituted with Glutathione S-transferase (GST), and weak induction was seen in E. coli BL21(DE3). The GST-FOXJ1 showed anomalous migration on denaturing gel electrophoresis (AM-DRE), migrating at approximately 83 kDa instead of its calculated molecular weight (Mr) of 72.4 kDa. It was also unstable and led to degradation products. The 6xHis tag was substituted with Glutathione S-transferase (GST) to improve induction and solubility. Codon-optimization improved the induction, but the protein still showed AM-DRE and instability. It seemed that the recombinant protein was either toxic or posed a metabolic burden to the E. coli cells or, once produced was prone to degradation due mainly to the lack of post-translational modification (PTM). This process is required for some eukaryotic proteins after they are manufactured in the ribosomal factory. Both the purified recombinant proteins exhibited cysteine-induced oligomerization via the formation of disulphide bridges since this was reduced using dithiothreitol (DTT). Both were equally functional as these individually bound to an oligonucleotide, a consensus DNA-binding sequence for FOX proteins. Further, the recombinant polypeptides corresponding to the C-terminus and N-terminus show anomalies indicating that the highly acidic residues (known as polyacidic gel-shifting domains) in these polypeptides contribute to the AM-DRE. We demonstrate for the first time that the recombinant HsFOXJ1 and its DBD bind to DNA, its polyacidic gel-shifting domains are the reason for the AM-DRE, is unstable leading to degradation products, exhibits cysteine-induced oligomerization and harbours intrinsically disordered regions.

The heterogeneous expression, extraction, and purification of recombinant Caldanaerobacter subterraneus subsp. tengcongensis apurine/apyrimidine endonuclease in Escherichia coli

Thermostable apurinic/apyrimidinic (AP) endonuclease (TtAP), cloned from Caldanaerobacter subterraneus subsp. tengcongensis, is an exonuclease III (Exo III) family protein with high-heat resistance, has activities of AP site endonuclease, 3′–5′ exonuclease, and 3′-nuclease, and facilitates efficient amplification of lengthy DNA fragments in PCR. However, the research of the combinant TtAP in Escherichia coli with its expression, large-scale extraction and purification of its protein was limited. In this study, we optimized the codons of TtAP gene for expression in E. coli and constructed a fusion gene encoding TtAP with a 6His tag (TtAP-6His). TtAP-6His was put into vector pET-30a(+) to form the expression vector pET-30a(+)-TtAP-6His, and was then introduced into E. coli strain Rosetta (DE3). We established a systematic process for the extraction of TtAP protein using 5 liters of bacterial suspension, including the optimization of IPTG induction time (6 h), followed by protein extraction using enzymolysis buffers, the heat treatment of temperature (70 °C) with 60 min to remove impurity, precipitation with ammonium sulfate (55 %), protein purification with Ni-affinity chromatography, and the enzyme activities finally were determined. The purification yield of TtAP-6His ranged from 73.67 to 115.25 mg/L (47 KU/mg).

Green and Fast Synthesis of NiCo-MOF for Simultaneous Purification-Immobilization of Bienzyme to Catalyze the Synthesis of Ginsenoside Rh2.

Traditional metal-organic frameworks (MOFs) preparation is generally time-consuming, polluting, and lacking specificity for enzyme immobilization. This paper introduced a facile, rapid, and green method to produce three MOFs subsequently employed to purify and coimmobilize recombinant glycosyltransferase (UGT) and recombinant sucrose synthetase (SUSy) using histidine tag (His-tag) for the specific adsorption of Ni2+ and Co2+ from MOFs. This method simplified enzyme purification from crude extracts and enabled enzymes to be reused. The results demonstrated that NiCo-MOF exhibited a higher enzyme load (115.9 mg/g) than monometallic MOFs. Additionally, the NiCo-MOF@UGT&SUSy demonstrated excellent stability and efficiently produced the rare ginsenoside Rh2 by catalyzing a coupling reaction (95.6 μg/mL), solving the problem of the substrate cost of uridine diphosphate glucose (UDPG). The NiCo-MOF@UGT&SUSy retained 68.97% of the initial activity after 10 cycles. Finally, molecular docking studies elucidated the conversion mechanism of the target product Rh2. This technique is important in the industrialization of ginsenoside production and enzyme purification.

Recombinant bovine serum albumin domain II as bioreceptor for ochratoxin A capture

Established chromatographic techniques for mycotoxin control in foodstuffs require prior sample enrichment and clean-up, typically achieved using immunoaffinity columns (IACs). Bovine serum albumin (BSA) has recently emerged as a cost-effective alternative to antibodies used in IACs. This study aimed at exploring the BSA domain II (BDII), which houses the primary binding site for ochratoxin A (OTA), as a bioreceptor for OTA capture. Recombinant BDII (rBDII) was produced in soluble form by Escherichia coli Origami 2(DE3), fused to a His6 (HisBDII) or thioredoxin-His6 (TrxBDII) tag, with yields up to 19 ± 4.3 mg/Lculture in shake-flask. Fluorescence and circular dichroism (CD) spectroscopy revealed interaction of OTA with both rBDII variants, with estimated binding constants for OTA-HisBDII/TrxBDII complexes in the range of 5.7–9.3 × 104 M−1. CD also showed an α/β structure of rBDII variants, in opposition to the predominant α-helical structure of whole BSA, and slight increase in their α-helical content upon binding to OTA. TrxBDII immobilized on Ni-NTA resin successfully captured OTA from spiked samples at the optimum pH range of 6.5–7.0, allowing OTA extraction, clean-up, and enrichment from spiked white grape juice, with up to 84 ± 7.4 % recovery.

99mTc(I)-Labeled His-Tagged Proteins: Impact in the Development of Novel Imaging Probes and in Drug Discovery.

Technetium-99 m (99mTc) remains the cornerstone of nuclear medicine for single photon emission computed tomography (SPECT) due to its widespread availability and chemical and physical features. Its multiple oxidation states allow for the design and production of radiopharmaceuticals with versatile properties, namely in terms of pharmacokinetic profile. 99mTc(V) is the most common oxidation state, but 99mTc(I) gained traction after the pioneering work of Alberto and colleagues, which resulted in the introduction of the organometallic core fac-[99mTc(CO)3(H2O)3]+. This core is readily available from [99mTcO4]- and displays three labile water molecules that can be easily swapped for ligands with different denticity and/or donor atoms in aqueous environment. This makes it possible to radiolabel small molecules as well as high molecular weight molecules, such as antibodies or other proteins, while assuring biological activity. Direct radiolabelling of those proteins with fac-[99mTc(CO)3]+ under mild conditions is accomplished through incorporation of a polyhistidine tag (His-tag), a commonly used tag for purification of recombinant proteins. This review aims to address the direct radiolabelling of His-tagged macromolecules with fac-[99mTc(CO)3]+ for development of molecular imaging agents and the impact of this technology in the discovery and development of imaging and/or therapeutic agents towards clinical application.

Gold Nanoparticles-Based Colorimetric Immunoassay of Carcinoembryonic Antigen with Metal–Organic Framework to Load Quinones for Catalytic Oxidation of Cysteine

This work reported gold nanoparticles (AuNPs)-based colorimetric immunoassay with the Cu-based metal–organic framework (MOF) to load pyrroloquinoline quinone (PQQ) for the catalytic oxidation of cysteine. In this method, both Cu2+ and PQQ in the MOF could promote the oxidation of inducer cysteine by redox cycling, thus limiting the cysteine-induced aggregation of AuNPs and achieving dual signal amplification. Specifically, the recombinant carcinoembryonic antigen (CEA) targets were anchored on the MOF through the metal coordination interactions between the hexahistidine (His6) tag in CEA and the unsaturated Cu2+ sites in MOF. The CEA/PQQ-loaded MOF could be captured by the antibody-coated ELISA plate to catalyze the oxidation of cysteine. However, once the target CEA in the samples bound to the antibody immobilized on the plate surface, the attachment of CEA/PQQ-loaded MOF would be limited. Cysteine remaining in the solution would trigger the aggregation of AuNPs and cause a color change from red to blue. The target concentration was positively related to the aggregation and color change of AuNPs. The signal-on competitive plasmonic immunoassay exhibited a low detection limit with a linear range of 0.01–1 ng/mL. Note that most of the proteins in commercial ELISA kits are recombinant with a His6 tag in the N- or C-terminal, so the work could provide a sensitive plasmonic platform for the detection of biomarkers.

Deleterious Effects of Histidine Tagging to the SH3b Cell Wall-Binding Domain on Recombinant Endolysin Activity.

Natural and artificial endolysins exhibit bactericidal effects by destroying peptidoglycans in the cell wall of gram-positive bacteria and are usually composed of an N-terminal catalytic domain (CTD) and a C-terminal cell wall-binding domain (CBD). The structures and receptors of CBDs are variable, but bacterial Src homology 3 (SH3b) CBDs are prevalent among the natural endolysins of Staphylococcus aureus. Moreover, although recombinant endolysins with a C-terminal 6x histidine tag (His-tag) are often produced and convenient to purify, the deleterious effects of His-tags on antibacterial activity have not been evaluated thoroughly. Recently, we reported that the antibacterial activity of a commercial lysostaphin without a His-tag differed from that of cell-free lysostaphin with a C-terminal His-tag, and lysostaphin also contains a C-terminal SH3b CBD. In this study, we directly compared the effects of His-tags on the antibacterial activities of lysostaphin and several chimeric lysins possessing different SH3b CBDs. We confirmed that antibacterial activity decreased 16.0-32.0-fold after a His-tag was added to the SH3b CBD.

Human serum albumin regulates the ornithine carbamoyltransferase production of Streptococcus pyogenes CS24 in vitro

The M12 protein is the main virulence factor of Streptococcus pyogenes as a mediator in the bacteria’s adhesion to numerous human proteins, including human serum albumin (HSA) and immunoglobulin. Based on our previous study, the interaction of the M12 protein with HSA is speculated to influence the synthesis of the ornithine carbamoyltransferase 12 (OCTase12), which may attach human fibronectin, preventing the bacteria from being recognized as a foreign material, thus defending itself against the human immune system. The purpose of this study was to use an immunological method to examine the impact of HSA on OCTase12 synthesis. The methods of this study were conducted with the overproduction of OCTase 12 in Escherichia coli BL21 (DE3) plysS containing pEToctase12 gene fused with polyhistidine tag to purify the protein using affinity chromatography with nickel as the ligand. The purified protein was then characterized using dot blot and the influence of HSA was investigated using Western blot analyses. This investigation was conducted by cultivating the E. coli in conditions with and without HSA. The Western blot analysis using the antibody against total cell proteins of S. pyogenes CS24 indicated that OCTase12 production was increased by the presence of HSA. Therefore, this early finding showing that HSA may influence the expression of the octase 12 gene.

Light-Activated Molecular Purification (LAMP) of Recombinant Proteins.

The production of recombinant proteins has become a focal point in biotechnology, with potential applications in catalysis, therapeutics, and diagnostics. Before their application, these proteins undergo cumbersome downstream processing, including multiple resin-based chromatography steps (ion exchange or affinity-based) to isolate the protein of interest from host cell proteins, which are more abundant. These methods often involve (1) nonspecific binding of host cell proteins onto the resin, (2) a trial and error approach in determining elution conditions for the protein of interest, and (3) complex functionalization of the resin. These processes are also further supplemented with additional processing steps including buffer exchange through dialysis or desalting. Despite the prevalence and need for proteins, challenges persist in optimizing elution conditions and minimizing downstream processing steps, which contribute to the overall cost, impeding their translation into the market. To address these challenges, there has been a growing interest in stimuli-responsive purification systems, which allow for precise control and modulation of the purification process for protein recovery by altering their properties or behavior in response to specific external conditions, such as heat, light, or chemicals. We have developed a light-activated molecular purification (LAMP) system, a stimuli-responsive chromatography technique where the purification of recombinant proteins is triggered by light. We employed a photocleavable protein (PhoCl1) that binds specifically to Ni-NTA resin through a hexa-histidine tag at its N-terminus. We harnessed the ability of PhoCl1 to undergo photocleavage into two fragments for the development of LAMP. To demonstrate LAMP, the protein of interest (POI) is genetically fused to the C-terminus of PhoCl1. The exposure to 405 nm light (1.5 mW cm-2 for 12 h) results in the release of POI into the supernatant. We showcased the potential of LAMP by purifying highly charged green fluorescent proteins and an enzyme, riboflavin kinase. Our custom-built violet light LED setup achieved more than 50% light-induced photocleavage of the fusion constructs, resulting in the release of more than 30% of the POI into the supernatant, with the remainder retained within the resin. All the proteins purified using LAMP were more than 90% pure. Moreover, the comparison of the riboflavin kinase purified through LAMP and the traditional chromatography (Ni-NTA affinity method) revealed no significant changes in the activity levels. These highlight the broad potential of LAMP in providing a facile, yet robust stimuli-responsive protein purification technique, which leverages the potential of light to purify the proteins and overcome the limitations of current conventional chromatography systems.

Combining Protein Phase Separation and Bio-orthogonal Linking to Coimmobilize Enzymes for Cascade Biocatalysis.

The designed and ordered co-immobilization of multiple enzymes for vectorial biocatalysis is challenging. Here, a combination of protein phase separation and bioorthogonal linking is used to generate a zeolitic imidazole framework (ZIF-8) containing co-immobilized enzymes. Zn2+ ions induce the clustering of minimal protein modules, such as 6-His tag, proline-rich motif (PRM) and SRC homology 3 (SH3) domains, and allow for phase separation of the coupled aldoketoreductase (AKR) and alcohol dehydrogenase (ADH) at low concentrations. This is achieved by fusing SpyCatcher and PRM-SH3-6His peptide fragments to the C and N termini of AKR, respectively, and the SpyTag to ADH. Addition of 2-methylimidazole results in droplet formation and enables in situ spatial embedding the recombinant AKR and ADH to generate the cascade biocalysis system encapsulated in ZIF-8 (AAE@ZIF). In synthesizing (S)-1-(2-chlorophenyl) ethanol, ater 6 cycles, the yield can still reach 91%, with 99.99% enantiomeric excess (ee) value for each cycle. However, the yield could only reach 72.9% when traditionally encapsulated AKR and ADH in ZIF-8 are used. Thus, this work demonstrates that a combination of protein phase separation and bio-orthogonal linking enables the in situ creation of a stable and spatially organized bi-enzyme system with enhanced channeling effects in ZIF-8.

Signal-On Detection of Caspase-3 with Methylene Blue-Loaded Metal-Organic Frameworks as Signal Reporters.

In this work, we report on an electrochemical method for the signal-on detection of caspase-3 and the evaluation of apoptosis based on the biotinylation reaction and the signal amplification of methylene blue (MB)-loaded metal-organic frameworks (MOFs). Zr-based UiO-66-NH2 MOFs were used as the nanocarriers to load electroactive MB molecules. Recombinant hexahistidine (His6)-tagged streptavidin (rSA) was attached to the MOFs through the coordination interaction between the His6 tag in rSA and the metal ions on the surface of the MOFs. The acetylated peptide substrate Ac-GDEVDGGGPPPPC was immobilized on the gold electrode. In the presence of caspase-3, the peptide was specifically cleaved, leading to the release of the Ac-GDEVD sequence. A N-terminal amine group was generated and then biotinylated in the presence of biotin-NHS. Based on the strong interaction between rSA and biotin, rSA@MOF@MB was captured by the biotinylated peptide-modified electrode, producing a significantly amplified electrochemical signal. Caspase-3 was sensitively determined with a linear range from 0.1 to 25 pg/mL and a limit of detection down to 0.04 pg/mL. Further, the active caspase-3 in apoptosis inducer-treated HeLa cells was further quantified by this method. The proposed signal-on biosensor is compatible with the complex biological samples and shows great potential for apoptosis-related diagnosis and the screening of caspase-targeting drugs.

Expression, purification and application of a recombinant, membrane permeating version of the light chain of botulinum toxin B.

Botulinum neurotoxins (BoNTs) are valuable tools to unveil molecular mechanisms of exocytosis in neuronal and non-neuronal cells due to their peptidase activity on exocytic isoforms of SNARE proteins. They are produced by Clostridia as single-chain polypeptides that are proteolytically cleaved into light, catalytic domains covalently linked via disulfide bonds to heavy, targeting domains. This format of two subunits linked by disulfide bonds is required for the full neurotoxicity of BoNTs. We have generated a recombinant version of BoNT/B that consists of the light chain of the toxin fused to the protein transduction domain of the human immunodeficiency virus-1 (TAT peptide) and a hexahistidine tag. His6-TAT-BoNT/B-LC, expressed in Escherichia coli and purified by affinity chromatography, penetrated membranes and exhibited strong enzymatic activity, as evidenced by cleavage of the SNARE synaptobrevin from rat brain synaptosomes and human sperm cells. Proteolytic attack of synaptobrevin hindered exocytosis triggered by a calcium ionophore in the latter. The novel tool reported herein disrupts the function of a SNARE protein within minutes in cells that may or may not express the receptors for the BoNT/B heavy chain, and without the need for transient transfection or permeabilization.

C-terminal Poly-histidine Tags Alter Escherichia coli Polyphosphate Kinase Activity and Susceptibility to Inhibition

In Escherichia coli, many environmental stressors trigger polyphosphate (polyP) synthesis by polyphosphate kinase (PPK1), including heat, nutrient restriction, toxic compounds, and osmotic imbalances. PPK1 is essential for virulence in many pathogens and has been the target of multiple screens for small molecule inhibitors that might serve as new anti-virulence drugs. However, the mechanisms by which PPK1 activity and polyP synthesis are regulated are poorly understood. Our previous attempts to uncover PPK1 regulatory elements resulted in the discovery of PPK1* mutants, which accumulate more polyP in vivo, but do not produce more in vitro. In attempting to further characterize these mutant enzymes, we discovered that the most commonly-used PPK1 purification method – Ni-affinity chromatography using a C-terminal poly-histidine tag – altered intrinsic aspects of the PPK1 enzyme, including specific activity, oligomeric state, and kinetic values. We developed an alternative purification strategy using a C-terminal C-tag which did not have these effects. Using this strategy, we were able to demonstrate major differences in the in vitro response of PPK1 to 5-aminosalicylic acid, a known PPK1 inhibitor, and observed several key differences between the wild-type and PPK1* enzymes, including changes in oligomeric distribution, increased enzymatic activity, and increased resistance to both product (ADP) and substrate (ATP) inhibition, that help to explain their in vivo effects. Importantly, our results indicate that the C-terminal poly-histidine tag is inappropriate for purification of PPK1, and that any in vitro studies or inhibitor screens performed with such tags need to be reconsidered in that light.