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Clostridium botulinum neurotoxin type A (BoNT/A) is one of the most potent biotoxins ever known. Its entry into neurons could block vesicle exocytosis to abolish the release of neurotransmitters from nerve terminals, thus leading to muscle paralysis. Although there are so many peptides, antibodies and chemical compounds claimed to have anti-toxin activity, no drug is available in the clinical application except equine antitoxin serum. In the present work, a short peptide inhibitor RRGW of BoNT/A was firstly identified by computer-aided ligand-receptor binding simulation, then an RRGW derived peptide was rational designed based on the fragment of SNAP-25 (141–206 aa). Proteolytic assay showed that the anti-toxin activity of the RRGW derived peptide was much higher than that of RRGW. Digit abduction score assay demonstrated that the derived peptide delayed BoNT/A-induced muscle paralysis at a lower concentration by 20-fold than RRGW. The results supported that RRGW derived peptide can be a potential BoNT/A inhibitor candidate for further treating botulism. [ABSTRACT FROM AUTHOR]

Macrocycle peptides are promising constructs for imaging and inhibiting extracellular, and cell membrane proteins, but their use for targeting intracellular proteins is typically limited by poor cell penetration. We report the development of a cell-penetrant high-affinity peptide ligand targeted to the phosphorylated Ser474 epitope of the (active) Akt2 kinase. This peptide can function as an allosteric inhibitor, an immunoprecipitation reagent, and a live cell immunohistochemical staining reagent. Two cell penetrant stereoisomers were prepared and shown to exhibit similar target binding affinities and hydrophobic character but 2-3-fold different rates of cell penetration. Experimental and computational studies resolved that the ligands' difference in cell penetration could be assigned to their differential interactions with cholesterol in the membrane. These results expand the tool kit for designing new chiral-based cell-penetrant ligands. Macrocycle peptides are promising constructs for imaging and inhibiting extracellular and cell membrane proteins, but their use for targeting intracellular proteins is typically limited by poor cell penetration. Here, the authors report the development of a cell-penetrant high-affinity peptide ligand targeted to the phosphorylated Ser474 epitope of the (active) Akt2 kinase. [ABSTRACT FROM AUTHOR]

The production of recombinant proteins is one of the most significant achievements of biotechnology in the last century. These proteins are produced in the eukaryotic or prokaryotic heterologous hosts. By increasing the omics data especially related to different heterologous hosts as well as the presence of new amenable genetic engineering tools, we can artificially engineer heterologous hosts to produce recombinant proteins in sufficient quantities. Numerous recombinant proteins have been produced and applied in various industries, and the global recombinant proteins market size is expected to be cast to reach USD 2.4 billion by 2027. Therefore, identifying the weakness and strengths of heterologous hosts is critical to optimize the large-scale biosynthesis of recombinant proteins. E. coli is one of the popular hosts to produce recombinant proteins. Scientists reported some bottlenecks in this host, and due to the increasing demand for the production of recombinant proteins, there is an urgent need to improve this host. In this review, we first provide general information about the E. coli host and compare it with other hosts. In the next step, we describe the factors involved in the expression of the recombinant proteins in E. coli. Successful expression of recombinant proteins in E. coli requires a complete elucidation of these factors. Here, the characteristics of each factor will be fully described, and this information can help to improve the heterologous expression of recombinant proteins in E. coli. [ABSTRACT FROM AUTHOR]

Objective: To examine the influence of widely used protein affinity tags and the tobacco PR1a signal peptide (SP) on detection, purification and bioactivity analyses of the small oomycete apoplastic effector SCR96 in planta. Results: Through agroinfiltration, the phytotoxic effector SCR96 of Phytophthora cactorum was expressed in Nicotiana benthamiana leaf apoplast as a fusion protein carrying single affinity tag (His, HA or FLAG) at either C- or N-terminus. Leaf necrosis caused by different affinity-tagged SCR96 varied among tags and replicates. All of tagged proteins can be detected by antibodies against SCR96. All of SCR96 fusions except N-terminally fused 6His-tagged protein were detected using tag antibodies, indicating that 6His tag may be degraded when fused at N-terminus. Interestingly, C-terminal His- and FLAG-tagged SCR96 maintained the biological activity after purification. In the substitution assay of SCR96 SP, we observed that PR1a SP can lead chimeric SCR96 expression in N. benthamiana, but the replacement totally disrupted its bioactivity. Conclusion: C-terminal His or FLAG tag, along with its original SP, is efficient enough to enable detection and purification of functional SCR96 from N. benthamiana leaf apoplast, which would facilitate plant–pathogen interaction studies. [ABSTRACT FROM AUTHOR]

We have created the Penn State Protein Ladder system to produce protein molecular weight markers easily and inexpensively (less than a penny a lane). The system includes plasmids which express 10, 15, 20, 30, 40, 50, 60, 80 and 100 kD proteins in E. coli. Each protein migrates appropriately on SDS-PAGE gels, is expressed at very high levels (10–50 mg per liter of culture), is easy to purify via histidine tags and can be detected directly on Western blots via engineered immunoglobulin binding domains. We have also constructed plasmids to express 150 and 250 kD proteins. For more efficient production, we have created two polycistronic expression vectors which coexpress the 10, 30, 50, 100 kD proteins or the 20, 40, 60, 80 kD proteins. 50 ml of culture is sufficient to produce 20,000 lanes of individual ladder protein or 3750 lanes of each set of coexpressed ladder proteins. These Penn State Protein Ladder expression plasmids also constitute useful reagents for teaching laboratories to demonstrate recombinant expression in E. coli and affinity protein purification, and to research laboratories desiring positive controls for recombinant protein expression and purification. [ABSTRACT FROM AUTHOR]

Modifications of the sidechain and backbone of peptides and proteins are commonly used to improve their stability, to enhance their biological function, to enable and improve imaging and detection capabilities via conjugation to imaging agents and to otherwise introduce new properties. The most common approaches for modification and conjugation typically target amino acids such as lysine and cysteine. However, less abundant residues such as tyrosine, methionine and tryptophan are also potential targets for orthogonal modification. Non‐traditional modifications of common amino acids such as cysteine can also be used to introduce novel functional groups in a peptide sidechain. Additionally, modification via sortase is also very precise and site specific in biological conditions. This mini‐review article describes recent advances in selective modification reactions of peptides and proteins via these less common approaches. © 2021 Society of Industrial Chemistry. [ABSTRACT FROM AUTHOR]

A glucose (Glu) sensor was designed by introducing synthetic cyclic peptides (CPs) as recognition receptors and Au nanoparticles assisted graphitic carbon nitride (AuNPs/g-C3N4) for electrochemiluminescence (ECL) enhancement. The synthetic CP receptor (cyclo-[−CNDNHCRDNDC−]) with natural active center of Glu binding protein can mimic the interactions between Glu and Glu binding protein to specifically capture Glu. The AuNPs were reduced on g-C3N4 and formed a new nanohybrid that can be applied as an ECL emitter. The AuNPs/g-C3N4 effectively ameliorated the ECL response of bare g-C3N4. The ECL enhancement mechanism was theoretically speculated through computer simulation. Glu quantification was conducted by recording ECL shifts induced by the binding of Glu to CPs. The linear detection range of the fabricated CPs-based ECL sensor was 1 to 100 mmol L−1, and the detection limit (LOD) was 0.57 nmol L−1 (S / N = 3). The CP-based ECL sensor also showed good specificity, repeatability, stability, and favorable recoveries in sample analysis. This work offer a promising analytical method for Glu assay in clinical diagnostics and bioprocess monitoring. [ABSTRACT FROM AUTHOR]

Plant virus-based expression systems are an alternative expression platform for the production of clinically and industrially useful recombinant proteins. Nonetheless, due to a lack of viral vector with the commercial potentials, it is urgent to design and develop new, versatile, and efficient plant virus vectors. The genome of Tomato bushy stunt virus (TBSV) offers an attractive alternative to being modified as a vector for producing heterologous proteins in plants. Here, we developed a set of novel fusion and non-fusion TBSV-CP replacement vectors, which provide more flexible and efficient tools for expressing proteins of interest in plants. An alternative tobacco plant, Nicotiana excelsiana, was used in this study as a host for newly constructed TBSV vectors because the unwanted necrotic effects were reported on the commonly used Nicotiana benthamiana host associated with expression of TBSV-encoded P19 protein. The data showed that TBSV vectors caused a symptomless infection and overexpressed reporter gene in N. excelsiana leaves, demonstrating that N. excelsiana is an ideal host plant for TBSV-mediated heterologous gene expression. Moreover, a TBSV non-fusion vector, dAUG, shows the similar accumulation level of reporter proteins to that of TMV- and PVX-based vectors in side-by-side comparison and provides more flexible aspects than the previously developed TBSV vectors. Collectively, our newly developed TBSV expression system adds a new member to the family of plant viral expression vectors and meanwhile offers a flexible and highly effective approach for producing proteins of interest in plants. Key points: • The TBSV-based transient expression system has been significantly improved. • The necrotic effects caused by viral P19 protein were avoided by the usage of N. excelsiana as a host plant. • The expression level of the non-fusion vector was similar to the most effective virus vectors reported so far. [ABSTRACT FROM AUTHOR]

In basic and applied biotechnology, design of affinity ligands has become essential for high‐capacity applications such as affinity‐based downstream processes for therapeutic molecules. Here, we established a proof‐of‐concept for the use of multimeric fusion single‐chain variable fragment (scFvs) as high‐capacity ligands in affinity adsorbents. Mono‐ and di/tri‐scFvs separated by Pro‐rich negatively charged linkers were designed, produced, and immobilized to 6% cross‐linked agarose beads. Frontal binding experiments with a target protein of 50 kDa resulted in up to 20 mg·mL−1 and 82% in dynamic binding capacity and utilization yield, respectively, at 100% breakthrough. The utilization of the binding sites was impacted by the ligand format and ligand density, rather than limitation in pore size of adsorbent as previously suggested. Overall, we demonstrated that multimeric fusion scFvs can successfully be developed and used as high‐capacity ligands in affinity adsorbents, enabling lean process design and alignment with process specifications. [ABSTRACT FROM AUTHOR]

The protein trap is a powerful tool for genetic and biochemical studies of gene function in the animal kingdom. Although the original protein trap was developed for flies, it can be easily adapted to other multicellular organisms, both known models and ones with an unsequenced genome. The protein trap has been successfully applied to the fruit fly, crustaceans Parhyale hawaiensis, zebrafish, and insect and animal cell cultures. This approach is based on the integration into genes of an artificial exon that carries DNA encoding a fluorescent marker, standardized immunoepitopes, an integrase docking site, and splice acceptor and donor sites. The protein trap for cell cultures additionally contains an antibiotic resistance gene, which facilitates the selection of trapped clones. Resulting chimeric tagged mRNAs can be interfered by dsRNA against GFP (iGFPi—in vivo GFP interference), or the chimeric proteins can be efficiently knocked down by deGradFP technology. Both RNA and protein knockdowns produce a strong loss of function phenotype in tagged cells. The fluorescent and protein affinity tags can be used for tagged protein localisation within the cell and for identifying their binding partners in their native complexes. Insertion into protein trap integrase docking sites allows the replacement of trap contents by any new constructs, including other markers, cell toxins, stop-codons, and binary expression systems such as GAL4/UAS, LexA/LexAop and QF/QUAS, that reliably reflect endogenous gene expression. A distinctive feature of the protein trap approach is that all manipulations with a gene or its product occur only in the endogenous locus, which cannot be achieved by any other method. [ABSTRACT FROM AUTHOR]

A Cu-free clickable polymer was synthesized and used to dip-coat plastic substrates to prepare Cu-free clickable surfaces. The surface density of dibenzocyclooctyne moieties was quantified using an azide-conjugated fluorophore and a conventional fluorometer, indicating that the clickable moieties displayed on a dip-coated surface were accessible from solutes in water. The present approach also succeeded in the control of the surface density of the clickable moieties. Azide-conjugated oligo DNA was immobilized on the surface using a Cu-free click reaction. [ABSTRACT FROM AUTHOR]

Introduction: Background: Neff regulatory protein as well as structural proteins such as MPER and V3 of HIV-1 is important targets in vaccine studies. Therefore, in the current study, the Nef-MPER-V3 fusion protein was cloned and expressed in prokaryotic expression system that will be evaluated as a candidate for future vaccine studies. Materials and Methods: The Nef-MPER-V3 sequence was synthesized and inserted into pET-28a(+) vector using Not I/HindIII enzymes. Then, the recombinant construct was expressed in BL21 (DE3) strain of E.coli. For optimization of protein expression, several factors such as time of induction, IPTG concentration and optical density (OD) were evaluated. The recombinant protein was purified on a Ni-NTA agarose column and analyzed by 12% SDS-PAGE. Finally, the fusion protein was confirmed by western blotting. Results: The target gene was successfully cloned into the recombinant vector. The best condition for the expression such as; 2 m IPTG, OD600 = 0.7 and growth 5 hours post-induction were determined and the recombinant protein was purified by affinity chromatography in denaturing condition with urea at a concentration of about 300 μg/ ml. The purified fusion protein migrated as a clear band of around 35 kDa in SDS-PAGE and was detectable using anti-Neff antibody in western blotting. Conclusion: Our results showed that the Nef-MPER-V3 protein was successfully expressed in prokaryotic system and purified protein may provide the antigen for future experiments for immunogenicity evaluation in mice is currently undertaken. [ABSTRACT FROM AUTHOR]

The influence of biotin and fluorescein tags attached to the N-terminus of peptides on their structural propensities is assessed by NMR methods. While the small peptides investigated are highly mobile with no uniquely preferred conformation, the introduction of the tags, in particular hydrophobic ones, clearly shows an influence on NMR parameters such as chemical shifts and relaxation properties, which are not restricted to the nearby residues, but also affect distant parts. Thus, long-range effects on structural propensities become evident and are cause for concern with respect to the interpretation of weak interaction tests, which rely upon the assumption that tags do not exert influence on intermolecular interactions. [ABSTRACT FROM AUTHOR]

Immobilized metal affinity chromatography (IMAC) is a specific high-capacity technique used in large-scale purification of proteins. IMAC exploits the ability of immobilized metal ions to form coordination bonds with atoms in the side chains of certain amino acids. The technique is generally robust. However, several factors still affect column binding capacity, retention, yield and purity of proteins during IMAC. It was observed that the recovery of 6× histidine, (His)6-tagged proteins from metal affinity columns differ significantly depending on the size of the protein. To test this observation, we determined the effect of protein size, flow-rate, number and position of (His)6 tag on the retention of highly expressing proteins on commercial Ni2+ and Co2+ IMAC columns. All experiments were performed in phosphate buffer to eliminate interference of amine-containing buffers with the binding of the (His)6 tag to the columns. Column retention was determined as the ratio of protein of interest in the supernatant (input) to flow-through (output). Data obtained suggest that regardless of the flow-rate, (His)6 tag position and number, the size of protein is a major factor affecting column retention and therefore recovery during column IMAC purification. Small and medium-sized proteins (~ 50 kDa) have higher column retention than bigger proteins, resulting in higher recovery. These outcomes provide important information to consider when performing IMAC. [ABSTRACT FROM AUTHOR]

The human tumor suppressor SMARCB1/INI1/SNF5/BAF47 (SNF5) is a core subunit of the multi-subunit ATP-dependent chromatin remodeling complex SWI/SNF, also known as Brahma/Brahma-related gene 1 (BRM/BRG1)-associated factor (BAF). Experimental studies of SWI/SNF are currently considerably limited by the low cellular abundance of this complex; thus, recombinant protein production represents a key to obtain the SWI/SNF proteins for molecular and structural studies. While the expression of mammalian proteins in bacteria is often difficult, the baculovirus/insect cell expression system can overcome limitations of prokaryotic expression systems and facilitate the co-expression of multiple proteins. Here, we demonstrate that human full-length SNF5 tagged with a C-terminal 3 × FLAG can be expressed and purified from insect cell extracts in monomeric and dimeric forms. To this end, we constructed a set of donor and acceptor vectors for the expression of individual proteins and protein complexes in the baculovirus/insect cell expression system under the control of a polyhedrin (polh), p10, or a minimal Drosophila melanogaster Hsp70 promoter. We show that the SNF5 expression level could be modulated by the selection of the promoter used to control expression. The vector set also comprises vectors that encode a 3 × FLAG tag, Twin-Strep tag, or CBP-3 × FLAG-TEV-ProteinA triple tag to facilitate affinity selection and detection. By gel filtration and split-ubiquitin assays, we show that human full-length SNF5 has the ability to self-interact. Overall, the toolbox developed herein offers the possibility to flexibly select the promoter strength as well as the affinity tag and is suggested to advance the recombinant expression of chromatin remodeling factors and other challenging proteins. [ABSTRACT FROM AUTHOR]

Background: Thrombopoietin (TPO) is an important cytokine that has a critical role in regulating hematopoietic stem cells (HSCs) proliferation and megakaryocyte differentiation. Because of scares amount of this protein in human plasma, in many biotechnological centers around the world, recombinant production of this protein has been carried out. This study was aiming to gene cloning and expression of recombinant thrombopoietin. Methods: This research is an experimental laboratory study carried out in Blood Transfusion Research Center, Tehran, Iran, from July 2016 to August 2017. At the beginning HepG2 cell line was cultured and RNA extraction was performed. Extracted RNA was used as template for cDNA synthesis and subsequently the synthesized cDNA was adopted to isolate TPO gene through polymerase chain reaction (PCR) reaction using designed primers. After isolating the TPO sequence from HepG2 cell line, the designated sequence was inserted into pET32 vectors. Recombinant plasmid was amplified by meriting from DH5α replicating system. The amplified plasmids were sequenced via chain termination method. Next step was transforming the recombinant plasmid into Rosetta-gami bacteria to express the recombinant protein. In order to induce protein expression, an appropriate amount of isopropyl β-D-1- thiogalactopyranoside (IPTG) was added to growth media, then bacterial lysate of expression host was prepared and assayed via polyacrylamide gel electrophoresis and western blotting test. Results: After sequencing of recombinant plasmid, it was confirmed that TPO sequence has been successfully colonized in adopted vector. Subsequent to induction of recombinant protein, total cell protein analysis affirmed that recombinant protein has been expressed in its soluble form at cytoplasmic condition. Location of expected recombinant protein band on polyacrylamide gel and reaction of recombinant protein with His-tag monoclonal antibody at western blotting was asserting that expressed protein is the one of interest. Conclusion: Rosetta-gami bacteria has capability of expressing recombinant thrombopoietin in its soluble form. By harnessing this method of recombinant protein expression, it would be possible to take advantage of high throughout bacterial expression system which would not produce inclusion body and its product doesn’t need further processing and refolding. [ABSTRACT FROM AUTHOR]

Emerging infectious diseases remain among the leading causes of global mortality. Traditional laboratory diagnostic approaches designed to detect and track infectious disease agents provide a framework for surveillance of bio threats. However, surveillance and outbreak investigations using such time-consuming approaches for early detection of pathogens remain the major pitfall. Hence, reasonable real-time surveillance systems to anticipate threats to public health and environment are critical for identifying specific aetiologies and preventing the global spread of infectious disease. The current review discusses the growing need for monitoring and surveillance of pathogens with the same zeal and approach as adopted by microbial forensics laboratories, and further strengthening it by integrating with the innovative nanotechnology for rapid detection of microbial pathogens. Such innovative diagnostics platforms will help to track pathogens from high risk areas and environment by pre-emptive approach that will minimize damages. The various scenarios with the examples are discussed where the high risk associated human pathogens in particular were successfully detected using various nanotechnology approaches with potential future prospects in the field of microbial forensics. [ABSTRACT FROM AUTHOR]

To develop an efficient protein expression system, we designed a late embryogenesis abundant (LEA) peptide by mutating the LEA peptide constructed in our previous study (LEA-I). The peptide is based on the repeating units of an 11mer motif characteristic of LEA proteins from Polypedilum vanderplanki larvae. In the amino acid sequence of the 13mer LEA peptide, glycine at the 6th and 12th positions was replaced with other amino acids via point mutations. Glutamic acid, lysine, leucine, and asparagine in the LEA peptide at the 6th and 12th positions increased green fluorescence protein (GFP) expression. The GFP expression of the mutated LEA peptide was 1.5 to 2.0 times higher than that without LEA peptide. In contrast, the serine-containing mutated LEA peptide has low GFP expression levels. We hypothesize that the position of amino acids and the nature of amino acid in LEA peptide are important for our coexpression system. These data suggest that the size, structure, and charge of amino acids in the LEA peptide improve the protection and expression of the target protein. The amino acid balance also plays an important role in the expression of the target protein. [ABSTRACT FROM AUTHOR]

The gram-positive bacterium Lactococcus lactis is a useful host for extracellular protein production. A main advantage of L. lactis over other bacterial expression systems is that lactococcal cells display low levels of autolysis and proteolysis. Previously, we developed a set of vectors for nisin-inducible extracellular production of N- or C-terminally hexa-histidine (His)-tagged proteins. The present study was aimed at expanding our portfolio of L. lactis expression vectors for protein purification and site-specific labeling. Specifically, we present two new groups of vectors allowing N- or C-terminal provision of proteins with a Strep-tag II or AVI-tag. Vectors for AVI-tagging encode an additional His-tag for protein purification. Another set of vectors allows removal of N-terminal Strep- or His-tags from expressed proteins with the tobacco etch virus protease. Two possible applications of the developed vectors are presented. First, we show that Strep-tagged LytM of Staphylococcus aureus in the growth medium of L. lactis can be directly bound to microtiter plates coated with an affinity reagent and used for enzyme-linked immunosorbent assays. Second, we show that the AVI-tagged Sle1 protein from S. aureus produced in L. lactis can be directly biotinylated and fluorescently labeled. The fluorescently labeled Sle1 was successfully applied for S. aureus re-binding studies, allowing subcellular localization by fluorescence microscopy. In conclusion, we have developed a set of expression vectors that enhances the versatility of L. lactis as a system for production of proteins with tags that can be used for affinity purification and site-specific protein labeling. [ABSTRACT FROM AUTHOR]

Affinity tags are vital tools for the production of high-throughput recombinant proteins. Several affinity tags, such as the hexahistidine tag, maltose-binding protein, streptavidin-binding peptide tag, calmodulin-binding peptide, c-Myc tag, glutathione S-transferase and FLAG tag, have been introduced for recombinant protein production. The fragment crystallizable (Fc) domain of the IgG1 antibody is one of the useful affinity tags that can facilitate detection, purification and localization of proteins and can improve the immunogenicity, modulatory effects, physicochemical and pharmaceutical properties of proteins. Fcγ recombinant forms a group of recombinant proteins called Fc-fusion proteins (FFPs). FFPs are widely used in drug discovery, drug delivery, vaccine design and experimental research on receptor–ligand interactions. These fusion proteins have become successful alternatives to monoclonal antibodies for drug developments. In this review, the physicochemical, biochemical, immunological, pharmaceutical and therapeutic properties of recombinant FFPs were discussed as a new generation of bioengineering strategies. [ABSTRACT FROM AUTHOR]

The rise of CuI-catalyzed click chemistry has initiated an increased demand for azido and alkyne derivatives of amino acid as precursors for the synthesis of clicked peptides. However, the use of azido and alkyne amino acids in peptide chemistry is complicated by their high cost. For this reason, we investigated the possibility of the in-house preparation of a set of five Fmoc azido amino acids: β-azido l-alanine and d-alanine, γ-azido l-homoalanine, δ-azido l-ornithine and ω-azido l-lysine. We investigated several reaction pathways described in the literature, suggested several improvements and proposed several alternative routes for the synthesis of these compounds in high purity. Here, we demonstrate that multigram quantities of these Fmoc azido amino acids can be prepared within a week or two and at user-friendly costs. We also incorporated these azido amino acids into several model tripeptides, and we observed the formation of a new elimination product of the azido moiety upon conditions of prolonged couplings with 2-(1 H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate/DIPEA. We hope that our detailed synthetic protocols will inspire some peptide chemists to prepare these Fmoc azido acids in their laboratories and will assist them in avoiding the too extensive costs of azidopeptide syntheses. Experimental procedures and/or analytical data for compounds 3- 5, 20, 25, 26, 30 and 43- 47 are provided in the supporting information. © 2017 The Authors Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd. [ABSTRACT FROM AUTHOR]

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

Some epigenetic modifier proteins have become validated clinical targets. With a few small molecule inhibitors already approved by national health administrations and many more in the pharmaceutical industry pipelines, there is a need for technologies that can promote full comprehension of the molecular action of these drugs. Proteomics, with its relatively unbiased nature, can contribute to a thorough understanding of the complexity of the megadalton complexes, which write, read and erase the histone code, and it can help study the on-target and off-target effect of the drugs designed to modulate their action. This review on the one hand gathers the published affinity probes able to decipher small molecule targets and off-targets in a close-to-native environment. These are small molecule analogues of epigenetic drugs conceived as protein target enrichment tools after they have engaged them in cells or lysates. Such probes, which have been designed for deacetylases, bromodomains, demethylases, and methyltransferases not only enrich their direct protein targets but also their stable interactors, which can be identified by mass spectrometry. Hence, they constitute a tool to study the epigenetic complexes together with other techniques also reviewed here: immunoaffinity purification with antibodies against native protein complex constituents or epitope tags, affinity matrices designed to bind recombinantly tagged protein, and enrichment of the complexes using histone tail peptides as baits. We expect that this toolbox will be adopted by more and more researchers willing to harness the spectacular advances in mass spectrometry to the epigenetic field. [ABSTRACT FROM AUTHOR]

The existing health care systems focus on treating diseases rather than preventing them. Patients are generally not tested unless physiological symptoms are appeared. When they do get tested, the results often take several days and can be inconclusive if the disease is at an early stage. In order to facilitate the diagnostics process and make tests more readily available for patients, the concept of "point of care testing" (POCT) has been brought up and developed in recent years. Field effect transistors (FET) using nanomaterial as a kind of biosensors have shown great characteristics for detection of a wide range of biomolecules due to their label-free, real time and ultrasensitive properties. In this paper, first of all, the working principles of such devices and recent developments in fabrication methods and surface functionalization are stated, and then some current research trends in field-effect transistor nanobiosensors are highlighted. Eventually key advantages and challenges of FET-based nanobiosensors as POCT devices are discussed as well. [ABSTRACT FROM AUTHOR]

Abnormal signaling of the protein kinase Akt has been shown to contribute to human diseases such as diabetes and cancer, but Akt has proven to be a challenging target for drugging. Using iterative in situ click chemistry, we recently developed multiple protein-catalyzed capture (PCC) agents that allosterically modulate Akt enzymatic activity in a protein-based assay. Here, we utilize similar PCCs to exploit endogenous protein degradation pathways. We use the modularity of the anti-Akt PCCs to prepare proteolysis targeting chimeric molecules that are shown to promote the rapid degradation of Akt in live cancer cells. These novel proteolysis targeting chimeric molecules demonstrate that the epitope targeting selectivity of PCCs can be coupled with non-traditional drugging moieties to inhibit challenging targets. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Highlights An Akt-targeted PCC allosterically activates Akt kinase activity in cells, The anti-Akt PCC was functionalized with a degradation tag to create a novel PROTAC, Combining PCCs with degradation tags is a new strategy to create PROTACs, In Brief A protein-catalyzed capture agent (PCC) was developed against the C-terminal hydrophobic motif of Akt, a traditionally 'undruggable' site, and shown to be an allosteric activator. Functionalization of the PCC with a degradation tag led to a new anti-Akt PROTAC. [ABSTRACT FROM AUTHOR]

ABSTRACT The downstream processing of proteins remains the most significant cost in protein production, and is largely attributed to rigorous chromatographic purification protocols, where the stringency of purity for biopharmaceutical products sometimes exceeds 99%. With an ever burgeoning biotechnology market, there is a constant demand for alternative purification methodologies, to ameliorate the dependence on chromatography, while still adhering to regulatory concerns over product purity and safety. In this article, we present an up-to-date view of bioseparation, with emphasis on magnetic separation and its potential application in the field. Additionally, we discuss the economic and performance benefits of synthetic ligands, in the form of peptides and miniaturized antibody fragments, compared to full-length antibodies. We propose that adoption of synthetic affinity ligands coupled with magnetic adsorbents, will play an important role in enabling sustainable bioprocessing in the future. Biotechnol. Bioeng. 2016;113: 11-25. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Antibodies are indispensable tools in biochemical research and play an expanding role as therapeutics. While hybridoma technology is the dominant method for antibody production, phage display is an emerging technology. Here, we developed and employed a high-throughput pipeline that enables selection of antibodies against hundreds of antigens in parallel. Binding selections using a phage-displayed synthetic antigen-binding fragment (Fab) library against 110 human SH3 domains yielded hundreds of Fabs targeting 58 antigens. Affinity assays demonstrated that representative Fabs bind tightly and specifically to their targets. Furthermore, we developed an efficient affinity maturation strategy adaptable to high-throughput, which increased affinity dramatically but did not compromise specificity. Finally, we tested Fabs in common cell biology applications and confirmed recognition of the full-length antigen in immunoprecipitation, immunoblotting and immunofluorescence assays. In summary, we have established a rapid and robust high-throughput methodology that can be applied to generate highly functional and renewable antibodies targeting protein domains on a proteome-wide scale. [ABSTRACT FROM AUTHOR]

In this work, hydrophilic graphene nanosheets (GNS) and the related magnetic nanocomposites were prepared by first tethering alkyne-functionalized graphene oxide (GO) with azide-modified glucose followed by deposition of FeO nanoparticles on the functionalized GNS. For the preparation of nanohybrids, two approaches were designed: in the first one, after chlorination of GO with thionyl chloride, GO was reacted with propargyl alcohol and subsequently clicked with azide-glucoside. The resulted sweet GNS were easily dispersed in water and stable for 2 weeks. In the second approach, GO was functionalized with 3,4,5-tris(prop-2-yn-1-yloxy)benzoic acid via hydroxyl groups on its basal plane and finally treated with azide-glucoside. The obtained hydrophilic GNS were stable in water for 3 weeks. The results showed that both glucose-grafted GO sheets were reduced by sodium ascorbate during click-coupling reaction; this is one of the many advantages of click reaction. Finally, FeO nanoparticles were deposited on the sweet GNS and were superparamagnetic, responded quickly to an external magnetic field and exhibited efficient adsorption towards methylene blue, as a cationic dye. No leaching was observed even after a week of placing a magnet close to the vial containing the dispersion of magnetic sweet GNS. [ABSTRACT FROM AUTHOR]

Inhalation anthrax has a rapid progression and high fatality rate. Pathology and death from inhalation of Bacillus anthracis spores are attributed to the actions of secreted protein toxins. Protective antigen (PA) binds and imports the catalytic component lethal factor (LF), a zinc endoprotease, and edema factor (EF), an adenylyl cyclase, into susceptible cells. PA-LF is termed lethal toxin (LTx) and PA-EF, edema toxin. As the universal transporter for both toxins, PA is an important target for vaccination and immunotherapeutic intervention. However, its quantification has been limited to methods of relatively low analytic sensitivity. Quantification of LTx may be more clinically relevant than LF or PA alone because LTx is the toxic form that acts on cells. A method was developed for LTx-specific quantification in plasma using anti-PA IgG magnetic immunoprecipitation of PA and quantification of LF activity that co-purified with PA. The method was fast (<4 h total time to detection), sensitive at 0.033 ng/mL LTx in plasma for the fast analysis (0.0075 ng/mL LTx in plasma for an 18 h reaction), precise (6.3-9.9 % coefficient of variation), and accurate (0.1-12.7 %error; n ≥ 25). Diagnostic sensitivity was 100 % ( n = 27 animal/clinical cases). Diagnostic specificity was 100 % ( n = 141). LTx was detected post-antibiotic treatment in 6/6 treated rhesus macaques and 3/3 clinical cases of inhalation anthrax and as long as 8 days post-treatment. Over the course of infection in two rhesus macaques, LTx was first detected at 0.101 and 0.237 ng/mL at 36 h post-exposure and increased to 1147 and 12,107 ng/mL in late-stage anthrax. This demonstrated the importance of LTx as a diagnostic and therapeutic target. This method provides a sensitive, accurate tool for anthrax toxin detection and evaluation of PA-directed therapeutics. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Fusion of peptide-based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram-range amounts of proteins. IMAC-Ni(II) columns have become the natural partners of 6xHis-tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His-tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur-containing molecules. In this work, we evaluated two different cysteine- and histidine-containing six amino acid tags linked to the N-terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine-containing tagged GFPs were able to bind to IMAC-Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC-Ni(II) system reaches less than 20% recovery of the cysteine-containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC-Pd(II) yields a recovery of 45% with a purification factor of 13. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The cyanobacterial NADPH:plastoquinone oxidoreductase complex (NDH-1), that is related to Complex I of eubacteria and mitochondria, plays a pivotal role in respiration as well as in cyclic electron transfer (CET) around PSI and is involved in a unique carbon concentration mechanism (CCM). Despite many achievements in the past, the complex protein composition and the specific function of many subunits of the different NDH-1 species remain elusive. We have recently discovered in a NDH-1 preparation from Thermosynechococcus elongatus two novel single transmembrane peptides (NdhP, NdhQ) with molecular weights below 5 kDa. Here we show that NdhP is a unique component of the ∼450 kDa NDH-1L complex, that is involved in respiration and CET at high CO2 concentration, and not detectable in the NDH-1MS and NDH-1MS' complexes that play a role in carbon concentration. C-terminal fusion of NdhP with his-tagged superfolder GFP and the subsequent analysis of the purified complex by electron microscopy and single particle averaging revealed its localization in the NDH-1L specific distal unit of the NDH-1 complex, that is formed by the subunits NdhD1 and NdhF1. Moreover, NdhP is essential for NDH-1L formation, as this type of NDH-1 was not detectable in a ΔndhP::Km mutant. [ABSTRACT FROM AUTHOR]

N-myristoyltransferase (NMT) is an indispensible enzyme, which exists as two isoforms (NMT1 and NMT2) in humans and has proven roles in development of cancerous states. It is thus a target for novel anti-cancer drug design, but understanding of the biochemical and functional differences of these isozymes is not fully deciphered. A soluble expression under the T7 promoter for human NMT1 was achieved in E. coli BL21 (DE3) cells, devoid of any isopropyl β- d-1-thiogalactopyranoside-based induction. The identity of expressed protein was confirmed by matrix-assisted laser desorption ionization mass spectrometry peptide-fingerprint analysis and a two-step purification protocol yielded homogeneous enzyme. The intact mass of the purified protein was verified by electrospray ionization mass spectrometry and found to be in agreement with the theoretical mass (48.141 vs. 48.140 kDa). The fluorescence spectrophotometric analyses of the ligand binding and enzyme activity demonstrated that the recombinant form is functional. The yield of purified protein was ~8-10 mg/L culture (batch to batch variation) with a specific activity value of 18,500 ± 513 U/mg of protein under the experimental conditions used. The final verification of the myristoylation was demonstrated by mass spectrometry analysis of reaction product. The described approach could be readily adapted for production of human NMT1, with high yields of pure enzyme preparations, which should aid in downstream applications involving inhibitor design and structure-function studies of NMT's. [ABSTRACT FROM AUTHOR]

Tuberculosis is a leading cause of death in developing countries. Efforts are being made to both prevent its spread and improve curability rates. Understanding the biology of the bacteria causing the disease, Mycobacterium tuberculosis (M. tuberculosis), is thus vital. We have implemented improved screening methods for protein–protein interactions based on affinity purification followed by high-resolution mass spectrometry. This method can be efficiently applied to both medium- and high-throughput studies aiming to characterize protein–protein interaction networks of tubercle bacilli. Of the 4 tested epitopes FLAG, enhanced green fluorescent protein (eGFP), protein A and haemagglutinin, the eGFP tag was found to be most useful on account of its easily monitored expression and its ability to function as a simultaneous tool for subcellular localization studies. It presents a relatively low background with cost-effective purification. RNA polymerase subunit A (RpoA) was used as a model for investigation of a large protein complex. When used as bait, it co-purified with all remaining RNA polymerase core subunits as well as many accessory proteins. The amount of RpoA strongly correlated with the amount of quantification peptide used as part of the tagging system in this study (SH), making it applicable for semi-quantification studies. Interactions between the components of the RpoA-eGFP protein complex were further confirmed using protein cross-linking. Dynamic changes in the composition of protein complexes under induction of UV damage were observed when UvrA-eGFP expressing cells treated with UV light were used to co-purify UvrA interaction partners. [ABSTRACT FROM AUTHOR]

Most protein purification procedures include an affinity tag fused to either the N or C-terminal end of the protein of interest as well as a procedure for tag removal. Tag removal is not straightforward and especially tag removal from the C-terminal end is a challenge due to the characteristics of enzymes available for this purpose. In the present study, we demonstrate the utility of the divalent uranyl ion in a new procedure for protein purification and tag removal. By employment of a GFP (green florescence protein) recombinant protein we show that uranyl binding to a phosphorylated C-terminal tag enables target protein purification from an E. coli extract by immobilized uranyl affinity chromatography. Subsequently, the tag can be efficiently removed by UV-irradiation assisted uranyl photocleavage. We therefore suggest that the divalent uranyl ion (UO22+) may provide a dual function in protein purification and subsequent C-terminal tag removal procedures. [ABSTRACT FROM AUTHOR]

The boost protein expression has been done successfully by simple co-expression with a late embryogenesis abundant (LEA)-like peptide in Escherichia coli. Frequently, overexpression of a recombinant protein fails to provide an adequate yield. In the study, we developed a simple and efficient system for overexpressing transgenic proteins in bacteria by co-expression with an LEA-like peptide. The design of this peptide was based on part of the primary structure of an LEA protein that is known hydrophilic protein to suppress aggregation of other protein molecules. In our system, the expression of the target protein was increased remarkably by co-expression with an LEA-like peptide consisting of only 11 amino acid residues. This could provide a practical method for producing recombinant proteins efficiently. [ABSTRACT FROM AUTHOR]

We report on a method to improve in vitro diagnostic assays that detect immune response, with specific application to HIV-1. The inherent polyclonal diversity of the humoral immune response was addressed by using sequential in situ click chemistry to develop a cocktail of peptide-based capture agents, the components of which were raised against different, representative anti-HIV antibodies that bind to a conserved epitope of the HIV-1 envelope protein gp41. The cocktail was used to detect anti-HIV-1 antibodies from a panel of sera collected from HIV-positive patients, with improved signal-to-noise ratio relative to the gold standard commercial recombinant protein antigen. The capture agents were stable when stored as a powder for two months at temperatures close to 60oC. [ABSTRACT FROM AUTHOR]

At present, monitoring of air at the workplace, in urban environments, and on battlefields; exhaled air from medical patients; air in packaged food containers; and so forth can be accomplished with different types of analytical instruments. Vapor sensors have their niche in these measurements when an unobtrusive, low-power, and cost-sensitive technical solution is required. Unfortunately, existing vapor sensors often degrade their vapor-quantitation accuracy in the presence of high levels of interferences and cannot quantitate several components in complex gas mixtures. Thus, new sensing approaches with improved sensor selectivity are required. This technological task can be accomplished by the careful design of sensing materials with new performance properties and by coupling these materials with the suitable physical transducers. This review is focused on the assessment of the capabilities of bionanomaterials and bioinspired nanostructures for selective vapor sensing. We demonstrate that these sensing materials can operate with diverse transducers based on electrical, mechanical, and optical readout principles and can provide vapor-response selectivity previously unattainable by using other sensing materials. This ability for selective vapor sensing provides opportunities to significantly impact the major directions in development and application scenarios of vapor sensors. [ABSTRACT FROM AUTHOR]

Background: Here we describe a novel approach used to identify the constituents of protein complexes with high fidelity, using the integrin-associated scaffolding protein PINCH as a test case. PINCH is comprised of five LIM domains, zinc-finger protein interaction modules. In Drosophila melanogaster, PINCH has two known high-affinity binding partners—Integrin-linked kinase (ILK) that binds to LIM1 and Ras Suppressor 1 (RSU1) that binds to LIM5—but has been postulated to bind additional proteins as well. Results: To purify PINCH complexes, in parallel we fused different affinity tags (Protein A and Flag) to different locations within the PINCH sequence (N- and C-terminus). We expressed these tagged versions of PINCH both in cell culture (overexpressed in Drosophila S2 cell culture in the presence of endogenous PINCH) and in vivo (at native levels in Drosophila lacking endogenous PINCH). After affinity purification, we analyzed PINCH complexes by a novel 2D-gel electrophoresis analysis, iGEO (interactions by 2D Gel Electrophoresis Overlap), with mass spectrometric identification of individual spots of interest. iGEO allowed the identification of protein partners that associate with PINCH under two independent purification strategies, providing confidence in the significance of the interaction. Proteins identified by iGEO were validated against a highly inclusive list of candidate PINCH interacting proteins identified in previous analyses by MuDPIT mass spectrometry. Conclusions: The iGEO strategy confirmed a core complex comprised of PINCH, RSU1, ILK, and ILK binding partner Parvin. Our iGEO method also identified five novel protein partners that specifically interacted with PINCH in Drosophila S2 cell culture. Because of the improved reproducibility of 2D-GE methodology and the increasing affordability of the required labeling reagents, iGEO is a method that is accessible to most moderately well-equipped biological laboratories. The biochemical co-purifications inherent in iGEO allow for rapid and unambiguous identification of the constituents of protein complexes, without the need for extensive follow-up experiments. [ABSTRACT FROM AUTHOR]

Expression and purification of aggregation-prone and disulfide-containing proteins in Escherichia coli remains as a major hurdle for structural and functional analyses of high-value target proteins. Here, we present a novel gene-fusion strategy that greatly simplifies purification and refolding procedure at very low cost using a unique hyperacidic module derived from the human amyloid precursor protein. Fusion with this polypeptide (dubbed FATT for Flag-Acidic-Target Tag) results in near-complete soluble expression of variety of extracellular proteins, which can be directly refolded in the crude bacterial lysate and purified in one-step by anion exchange chromatography. Application of this system enabled preparation of functionally active extracellular enzymes and antibody fragments without the need for condition optimization. [ABSTRACT FROM AUTHOR]

Recombinant protein purification with affinity tags is a widely employed technique. One of the most common tags used for protein purification is the histidine tag (His). In this work, we use a tandem starch-binding domain (SBD) as a tag for protein purification. Four proteins from different sources were fused to the SBD, and the resulting fusion proteins were purified by affinity chromatography using the His or the SBD. The results showed that the SBD is superior to the His for protein purification. The efficient adsorption of the fusion proteins to raw corn starch was also demonstrated, and two fusions were selected to test purification directly using raw starch from rice, corn, potato, and barley. The two fusion proteins were successfully recovered from crude bacterial extract using raw starch, thus demonstrating that the SBD can be used as an efficient affinity tag for recombinant protein purification on an inexpensive matrix. [ABSTRACT FROM AUTHOR]

Background: Recombineering is a genetic engineering tool that enables facile modification of large episomal clones, e.g. BACs, fosmids. We have previously adapted this technology to generate, directly from fosmid-based genomic clones, fusion gene reporter constructs designed to investigate gene expression patterns in C. elegans. In our adaptation a rpsL-tet(A) positive/negative-selection cassette (RT-cassette) is first inserted and then, under negative selection, seamlessly replaced with the desired sequence. We report here on the generation and application of a resource comprising two sets of constructs designed to facilitate this particular recombineering approach. Results: Two complementary sets of constructs were generated. The first contains different fluorescent protein reporter coding sequences and derivatives while the second set of constructs, based in the copy-number inducible vector pCC1Fos, provide a resource designed to simplify RT-cassette-based recombineering. These latter constructs are used in pairs the first member of which provides a template for PCR-amplification of an RT-cassette while the second provides, as an excised restriction fragment, the desired fluorescent protein reporter sequence. As the RT-cassette is flanked by approximately 200 bp from the ends of the reporter sequence the subsequent negative selection replacement step is highly efficient. Furthermore, use of a restriction fragment minimizes artefacts negating the need for final clone sequencing. Utilizing this resource we generated single-, double- and triple-tagged fosmid-based reporters to investigate expression patterns of three C. elegans genes located on a single genomic clone. Conclusions: We describe the generation and application of a resource designed to facilitate counter-selection recombineering of fosmid-based C. elegans genomic clones. By choosing the appropriate pair of 'insertion' and 'replacement' constructs recombineered products, devoid of artefacts, are generated at high efficiency. Gene expression patterns for three genes located on the same genomic clone were investigated via a set of fosmid-based reporter constructs generated with the modified protocol. [ABSTRACT FROM AUTHOR]

In this review, we discuss a strategy to bring genomics and proteomics into single cells by super-resolution microscopy. The basis for this new approach are the following: given the 10 nm resolution of a super-resolution microscope and a typical cell with a size of (10 µm)3, individual cells contain effectively 109 super-resolution pixels or bits of information. Most eukaryotic cells have 104 genes and cellular abundances of 10–100 copies per transcript. Thus, under a super-resolution microscope, an individual cell has 1000 times more pixel volume or information capacities than is needed to encode all transcripts within that cell. Individual species of mRNA can be uniquely identified by labeling them each with a distinct combination of fluorophores by fluorescence in situ hybridization. With at least 15 fluorophores available in super-resolution, hundreds of genes in can be barcoded with a three-color barcode (3C15 = 455). These calculations suggest that by combining super-resolution microscopy and barcode labeling, single cells can be turned into informatics platforms denser than microarrays and that molecular species in individual cells can be profiled in a massively parallel fashion. [ABSTRACT FROM PUBLISHER]