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Recombinant protein production in Escherichia coli is a fundamental aspect of biotechnology. Fusion tags are commonly used to enhance solubility and facilitate purification. However, these tags can lead to challenges such as low yields, complicated purification processes, and the necessity for tag removal, especially when dealing with peptides. This study introduces a novel fusion tag called SmallTalk, a truncated version of the small metal‐binding protein SmbP. Weighing in at 5 kDa, SmallTalk includes two of the four α‐helices found in SmbP. It retains the ability to bind Ni(II) ions, which enables purification through IMAC. In this work, we assessed the efficiency of SmallTalk in expressing and purifying both a model protein, the green fluorescent protein, and the antimicrobial peptide Bin1b. Both proteins were effectively expressed and purified using IMAC, demonstrating SmallTalk's value as an affinity tag, yielding 7.2 mg·L−1 of cell culture for the green fluorescent protein and up to 9.8 mg·L−1 for Bin1b. Antimicrobial assays conducted with SmallTalk‐tagged Bin1b showed activity against Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, with minimum inhibitory concentrations ranging from 7.5 to 22.5 μm. Importantly, SmallTalk enabled the full retention of Bin1b's antimicrobial activity without the need for its removal, significantly simplifying the production process. These findings indicate that SmallTalk provides a promising strategy for the recombinant production of peptides. This tag has the potential to enhance the expression, purification, and functional analysis of antimicrobial peptides, which are increasingly being pursued as alternatives to antibiotics in the fight against antimicrobial resistance. The SmallTalk fusion tag allows for the efficient expression and purification of soluble recombinant proteins or peptides in Escherichia coli. Testing with SmallTalk‐GFP confirmed that the proteins were soluble and folded correctly, while SmallTalk‐Bin1b maintained its antimicrobial activity against various bacterial isolates. This streamlined workflow showcases the versatility of the SmallTalk system in producing functional, biologically active proteins and peptides.

For the study of amyloid beta (Aβ) associated toxicity which is supposed to be the main pathological agent in Alzheimer’s disease (AD), it is important to secure Aβ peptide with appropriate biological activity. However, commercial and synthetic Aβ often have some pitfalls like less cell toxicity, prompt aggregation and excess price, using recombinant technology, these issues can be resolved though the method also suffered from some problems such as low yield, aggregation and prolong time to purify. Thus, we previously developed an easy, economic and convenient method for Aβ42 purification using highly expressed GroES-Ubiquitin-Aβ42 fusion protein. The method was efficient, but further development was performed to improve the procedure and increase the yield. Focus was on the isolation of the fusion protein (GroES-Ubiquitin) from Aβ42 peptide. After a series of systematic testing with several chemicals, we found that methanol could precipitate efficiently the fusion protein, while the Aβ peptide was recovered in the supernatant. By this method, Aβ peptide was easily purified without tedious chromatographic steps which are main obstacles to purify the peptide in the previous method. This method yielded ~20 mg highly pure Aβ42 peptide from 1-liter bacterial culture. Different biophysical characterizations and bioactivity assays indicate that the peptide purified using this method was competitive with others which have been previously reported whereas considering the simplicity, final yield and time of purification, this method is the optimal solution.

Background Proteins with novel functions or advanced activities developed by various protein engineering techniques must have sufficient solubility to retain their bioactivity. However, inactive protein aggregates are frequently produced during heterologous protein expression in Escherichia coli . To prevent the formation of inclusion bodies, fusion tag technology has been commonly employed, owing to its good performance in soluble expression of target proteins, ease of application, and purification feasibility. Thus, researchers have continuously developed novel fusion tags to expand the expression capacity of high-value proteins in E. coli . Results A novel fusion tag comprising carbohydrate-binding module 66 (CBM66) was developed for the soluble expression of heterologous proteins in E. coli . The target protein solubilization capacity of the CBM66 tag was verified using seven proteins that are poorly expressed or form inclusion bodies in E. coli : four human-derived signaling polypeptides and three microbial enzymes. Compared to native proteins, CBM66-fused proteins exhibited improved solubility and high production titer. The protein-solubilizing effect of the CBM66 tag was compared with that of two commercial tags, maltose-binding protein and glutathione-S-transferase, using poly(ethylene terephthalate) hydrolase (PETase) as a model protein; CBM66 fusion resulted in a 3.7-fold higher expression amount of soluble PETase (approximately 370 mg/L) compared to fusion with the other commercial tags. The intact PETase was purified from the fusion protein upon serial treatment with enterokinase and affinity chromatography using levan-agarose resin. The bioactivity of the three proteins assessed was maintained even when the CBM66 tag was fused. Conclusions The use of the CBM66 tag to improve soluble protein expression facilitates the easy and economic production of high-value proteins in E. coli .

Antibiotic-resistant pathogens have become a great universal health concern. Antimicrobial peptides (AMPs) are small amphipathic and cationic polypeptides with high therapeutic potential against various microorganisms containing drug-resistant strains. Two major groups of these peptides, which have antibacterial activity against Gram-positive and Gram-negative bacteria, antiviral activity, and even antifungal activity, are defensins and cathelicidins. Hybridization of various AMPs is an appropriate approach to achieving new fusion AMPs with high antibacterial activity but low cellular toxicity. In the current research, the amino-acid sequence of human cathelicidin LL-37 (2-31) and Human beta-defensin (hBD)-129 were combined, and the fusion protein was evaluated by bioinformatics tool. The designed AMP gene sequence was commercially synthesized and cloned in the pET-28a expression vector. The LL-37/hBD-129 fusion protein was expressed in E.coli BL21-gold (DE3). The expression of the recombinant protein was evaluated using the SDS-PAGE method. The LL37/hBD-129 was successfully expressed as a recombinant hybrid AMP in E.coli BL21-gold (DE3) strain. Purification of the expressed AMP was performed by Ni–NTA column affinity chromatography, and the purified AMP was validated using the Western blot technic. Finally, the antimicrobial activity of the fusion AMP against Staphylococcus aureus and Escherichia coli bacteria was assessed. Based on the in silico analysis and experimental evaluations, the fusion AMP showed a significant antimicrobial effect on E. coli and Staphylococcus aureus bacteria.

Dermaseptins belonging to a large family of cationic membrane-disruption antimicrobial peptides display extensive antibacterial and antiproliferative activities depending on a coil-to-helix transition and the specific structural parameters. Herein, a novel dermaseptin peptide named Der-PS4 was discovered from the skin secretion of the waxy monkey tree frog, Phyllomedusa sauvagii. The complementary DNA (cDNA)-encoding precursor was obtained relying on “shotgun” cloning, and afterwards, a mature peptide amino acid sequence was identified by reverse-phase high performance liquid chromatography (RP-HPLC) and MS/MS. Specimens were chemically synthesized and applied for further functional studies. Structural analysis demonstrated a higher α-helical content in the membrane-mimetic environment compared with that in the ammonium acetate/water circumstance. Der-PS4 displayed a broad spectrum of antimicrobial activities against tested pathogenic microorganisms, however, exhibiting slight membrane-damaging effectiveness towards horse red blood cells. Coincident with the inhibitory activities on pathogens, Der-PS4 also showed considerable biofilm eradicating impact. Also, Der-PS4 penetrated cell membrane in a relative short period under each minimum bactericidal concentration. In addition, Der-PS4 possessed antiproliferative capacity against five cancer cell lines, while presenting slight suppressing effect on human microvascular endothelial, HMEC-1. These findings provide a promising insight for the discovery and development of novel drugs from a natural source.

Primary immune thrombocytopenia is an autoimmune disorder marked by accelerated platelet destruction and reduced production, posing risks of severe bleeding and mortality. Thrombopoietin (TPO) mimetic peptide (TMP) stimulates platelet generation via TPO receptor activation but is hindered by rapid clearance and short half-life. The primary objective of this study is to optimize TMP fusion proteins with the albumin-binding domain (ABD) for enhanced expression in Escherichia coli ( E. coli ), extended half-life via indirect FcRn-mediated recycling, and improved thrombopoietic activity in cellular assays and murine models. TMP dimers were fused to the C-terminus of ABD. GS peptides with either Cys or Ala were fused to the N-terminus of ABD (yielding C-ABD-2TMP and A-ABD-2TMP fusion proteins). The fusion proteins were expressed in E. coli BL21 (DE3) with Trx-tags for solubility, purified by Ni–NTA and ion exchange chromatography, and dimerized via disulfide bonds (2C-ABD-2TMP). Thermal stability was assessed by circular dichroism; HSA affinity by ELISA; bioactivity by MO7e cell proliferation assay; and in vivo pharmacokinetics and platelet stimulation in C57BL/6 mice. The fusion proteins were successfully expressed in E. coli . Following purification via Ni 2+ –NTA, tag cleavage, ion-exchange chromatography, and disulfide bond formation, high-purity fusion proteins were obtained: disulfide-bonded 2C-ABD-2TMP and A-ABD-TMP lacking disulfide bonds. Fusion proteins displayed highly stability up to 70 °C. 2C-ABD-2TMP exhibited an apparent EC 50 for HSA of 2.1 ± 0.6 nM (vs. 5.5 ± 1.1 nM for A-ABD-2TMP). In MO7e cells, 2C-ABD-2TMP promoted dose-dependent proliferation, exceeding 2TMP at 25 nM ( p  < 0.01) and A-ABD-2TMP ( p  < 0.05). In mice, 150 nmol/kg 2C-ABD-2TMP increased platelets to 3.1 × 10 9 /mL by day 12, sustained exceeding 2.4 × 10 9 /mL at day 18 (AUC p  < 0.01 vs. controls). The 2C-ABD-2TMP fusion protein displayed a half-life of 17.6 ± 2.9 h, while the A-ABD-2TMP exhibited a half-life of 13.2 ± 1.6 h. significantly longer than 2TMP alone (~ 1 h). ABD fusion enhanced the pharmacokinetics and thrombopoietic activity of 2TMP by enabling binding to HSA while retaining the ability to activate the TPO receptor.

EF-1 is a novel peptide derived from two bacteriocins, plantaricin E and plantaricin F. It has a strong antibacterial activity against Escherichia coli and with negligible hemolytic effect on red blood cells. However, the chemical synthesis of EF-1 is limited by its high cost. In this study, we established a heterologous expression of EF-1 in Pichia pastoris. The transgenic strain successfully expressed hybrid EF-1 peptide, which had a molecular weight of ~5 kDa as expected. The recombinant EF-1 was purified by Ni2+ affinity chromatography and reversed-phase high performance liquid chromatography (RP-HPLC), which achieved a yield of 32.65 mg/L with a purity of 94.9%. The purified EF-1 exhibited strong antimicrobial and bactericidal activities against both Gram-positive and -negative bacteria. Furthermore, propidium iodide staining and scanning electron microscopy revealed that EF-1 can directly induce cell membrane permeabilization of E. coli. Therefore, the hybrid EF-1 not only preserves the individual properties of the parent peptides, but also acquires the ability to disrupt Gram-negative bacterial membrane. Meanwhile, such an expression system can reduce both the time and cost for large-scale peptide production, which ensures its potential application at the industrial level.

The molecular complexity of mammalian proteomes demands new methods for mapping the organization of multiprotein complexes. Here, we combine mouse genetics and proteomics to characterize synapse protein complexes and interaction networks. New tandem affinity purification (TAP) tags were fused to the carboxyl terminus of PSD‐95 using gene targeting in mice. Homozygous mice showed no detectable abnormalities in PSD‐95 expression, subcellular localization or synaptic electrophysiological function. Analysis of multiprotein complexes purified under native conditions by mass spectrometry defined known and new interactors: 118 proteins comprising crucial functional components of synapses, including glutamate receptors, K + channels, scaffolding and signaling proteins, were recovered. Network clustering of protein interactions generated five connected clusters, with two clusters containing all the major ionotropic glutamate receptors and one cluster with voltage‐dependent K + channels. Annotation of clusters with human disease associations revealed that multiple disorders map to the network, with a significant correlation of schizophrenia within the glutamate receptor clusters. This targeted TAP tagging strategy is generally applicable to mammalian proteomics and systems biology approaches to disease. Synopsis Systems biology has the potential to explain physiological processes as emergent properties of sets of genes and proteins. Beyond simple cellular systems, the challenge of delivering systems biology into the intact and freely behaving animal will require new methods. Currently, the most widely used approach is immunoprecipitation of the target protein and its associate binding proteins. This method suffers from the drawbacks of single step purification strategies that include a high level of non‐specific background proteins amongst other limitations. To overcome these limitations we demonstrate that the tandem affinity purification (TAP) technology originally developed in yeast (Rigaut et al , 1999 ), when combined with gene targeting, can be used to efficiently isolate highly specific complexes from mouse. The ‘targeted TAP tagging’ strategy combines the two major advantages of each system. The first advantage is that the insertion of two tags into the protein of interest allows two consecutive purification steps that facilitate the recovery of protein complexes with high confidence and decreases the recovery of non‐specific proteins or weak interactors. The second advantage, conferred by targeting the endogenous gene, is that the tagged protein is expressed under its natural regulatory mechanisms. We have designed an endogenous TAP targeting strategy to isolate complexes from mouse brain excitatory synapses. The brain is the most complex organ from a cellular and molecular perspective and thus an ideal model to explore the TAP method. Post Synaptic Density 95 (PSD‐95/Dlg4) is an adaptor protein comprised of PDZ, SH3 and GK domains and is expressed in the postsynaptic terminal of excitatory synapses where it organizes signaling from neurotransmitter receptors to downstream pathways (Kornau et al , 1995 ; Hunt et al , 1996 ; Tu et al , 1999 ; Husi et al , 2000 ; Nehring et al , 2000 ; Dosemeci et al , 2007 ; Carlisle et al , 2008 ). Mice carrying a knockout mutation in PSD‐95 show it is essential for synaptic plasticity and a range of important behaviours (Migaud et al , 1998 ; El‐Husseini et al , 2000 ; Beique et al , 2006 ). Here, a new TAP tag was fused to the carboxyl terminus of PSD‐95 using gene targeting in mice. Homozygous mice showed no detectable abnormalities in PSD‐95 expression, subcellular localization or synaptic electrophysiological function (Figure 2 ). As a result of four independent tandem purifications and mass spectrometry analysis, we were able to define PSD‐95 core complexes with high sensitivity and reproducibility. The four purifications show an average of 125±19 proteins, having 118 proteins (94%) common in at least three of four replicates. This reproducibility rate is among the highest rate reported for systematic protein complex isolation. To further validate this interaction data we compared it to information from public datasets. Of the 118 proteins, 22% were proteins that directly bind PSD‐95 and 18% were proteins not previously found in other PSD‐95 analysis. All together, these data show robust reproducibility and sensitivity of this method for purifying synaptic complexes. These PSD‐95 core complexes comprise key functional components of synapses including the glutamate neurotransmitter receptors, K + channels, scaffolding and signaling proteins. These complexes contain ionotropic glutamate receptors of the NMDA, AMPA and kainate subtypes as well as major K + channels that together are the major postsynaptic constituents responsible for synaptic transmission and shaping the postsynaptic electrophysiological response to presynaptic input (Watanabe et al , 2002 ; Chen et al , 2006 ; Kim et al , 2007 ). We believe that this is the first method that has allowed the robust copurification of these proteins. To explore functional organization using network models, we manually curated interactions (Pocklington et al , 2006 ) and the UniHi database ( http://www.mdc‐berlin.de/unihi ) to identify 119 interactions between 50 proteins (excluding self‐interactions) of the PSD‐95 core complexes. Network clustering of the interacting proteins showed 40 out of the 50 proteins formed a large connected component (major connected component, MCC) and a modular structure that was segregated into 5 clusters referred to as cluster a (Cla) to cluster e (Cle) (Figure 5A ). In addition to the 5 MCC clusters, 2 further disconnected clusters (‘Clf’ and ‘Clg’) were found. Of great interest is the location and proximity of the receptors and channels responsible for the postsynaptic depolarization and subsequent action potential generation. All NMDA, AMPA and kainate glutamate receptors were restricted to Cla and Clb and the voltage‐dependent K + channels were found in Cla and Clc (entirely comprised of K + channels). It therefore appears that Cla, Clb and Clc are enriched with membrane proteins responsible for electrical properties of the postsynaptic terminal. The central role of PSD‐95 was supported by calculation of the shortest path from each protein to every other protein and PSD‐95 showed the lowest. Annotation of clusters with human disease associations revealed that multiple disorders map onto the network with a highly significant correlation of schizophrenia within the glutamate receptor clusters ( P <10−6). 20 genes involved in schizophrenia were significantly associated with the clusters Cla and Clb that contains all the glutamate receptors and MAGUK/Dlg proteins (Figure 5B ). Mapping the primary interactors of these schizophrenia proteins recruited many other proteins found in the other modules of the network. This suggests that the overall network and its different modules are a substrate for schizophrenia, and not simply the glutamate receptors, as was generally considered in the ‘glutamate hypothesis’ of schizophrenia (Greene, 2001 ; Coyle, 2006 ; Lisman et al , 2008 ). This targeted TAP tagging strategy is generally applicable to mammalian proteomics and systems biology approaches to disease. TAP tagged mice are a valuable resource and useful for a wide range of physiological studies and whole animal studies. A novel approach for isolating native protein complexes from mouse tissues using gene targeting of tandem affinity tags is presented. A protein core complex from brain synapses comprising principal electrophysiological and signalling components for synaptic transmission and synaptic plasticity was isolated. The protein interaction network shows clusters of functionally distinct proteins and schizophrenia susceptibility genes. This targeted TAP tagging method has general application to all types of protein complexes in the mouse and will be particularly useful for analysing molecular networks and systems biology in the intact animal.

NZ2114, a new variant of plectasin, was overexpressed in Pichia pastoris X-33 via pPICZαA for the first time. The total secreted protein of fermentation supernatant reached 2,390 mg/l (29 °C) and 2,310 mg/l (25 °C), and the recombinant NZ2114 (rNZ2114) reached 860 mg/l (29 °C) and 1,309 mg/l (25 °C) at 96 h induction in a 5-l fermentor, respectively.The rNZ2114 was purified by cation exchange chromatography, and its yield was 583 mg/l with 94.8 % purity. The minimal inhibitory concentration (MIC) of rNZ2114 to four ATCC strains of Staphyloccocus aureus was evaluated from 0.028 to 0.90 μM. Meanwhile, it showed potent activity (0.11–0.90 μM) to 20 clinical isolates of MRSA. The rNZ2114 killed over 99.9 % of tested S. aureus (ATCC 25923 and ATCC 43300) in Mueller-Hinton medium within 6 h when treated with 4 × MIC. The postantibiotic effect of rNZ2114 to S. aureus ATCC 25923 and ATCC 43300 was 18.6–45.6 and 1.7–3.5 h under 1×, 2×, and 4× MIC, respectively. The fractional inhibitory concentration index (FICI) indicated a synergistic effect between rNZ2114 and kanamycin, streptomycin, and vancomycin against S. aureus ATCC 25923 (FICI = 0.125), and additivity between rNZ2114 and ampicillin, spectinomycin (FICI = 0.625), respectively. To S. aureus ATCC 43300 [methicillin-resistant S. aureus (MRSA)], rNZ2114 showed a synergistic effect (FICI = 0.125–0.3125) with kanamycin, ampicillin, streptomycin, and vancomycin, and antagonism with spectinomycin (FICI = 8.0625). The rNZ2114 caused only less than 0.1 % hemolytic activity in the concentration of 128 μg/ml, and showed a good thermostability from 20 to 80 °C. In addition, it exhibited the highest activity at pH 8.0. These results suggested that large-scale production of NZ2114 is feasible using the P. pastoris expression system, and it could be a new potential antimicrobial agent for the prevention and treatment of S. aureus especially for MRSA infections.

A major bottleneck in structural, biochemical and biophysical studies of proteins is the need for large amounts of pure homogenous material, which is generally obtained by recombinant overexpression. Here we introduce a vector collection, the pCri System, for cytoplasmic and periplasmic/extracellular expression of heterologous proteins that allows the simultaneous assessment of prokaryotic and eukaryotic host cells (Escherichia coli, Bacillus subtilis, and Pichia pastoris). By using a single polymerase chain reaction product, genes of interest can be directionally cloned in all vectors within four different rare restriction sites at the 5'end and multiple cloning sites at the 3'end. In this way, a number of different fusion tags but also signal peptides can be incorporated at the N- and C-terminus of proteins, facilitating their expression, solubility and subsequent detection and purification. Fusion tags can be efficiently removed by treatment with site-specific peptidases, such as tobacco etch virus proteinase, thrombin, or sentrin specific peptidase 1, which leave only a few extra residues at the N-terminus of the protein. The combination of different expression systems in concert with the cloning approach in vectors that can fuse various tags makes the pCri System a valuable tool for high throughput studies.