A novel protein fusion partner, carbohydrate-binding module family 66, to enhance heterologous protein expression in Escherichia coli.

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.

Distribution of the Water-Soluble Astaxanthin Binding Carotenoprotein (AstaP) in Scenedesmaceae.

Photooxidative stress-inducible water-soluble astaxanthin-binding proteins, designated as AstaP, were identified in two Scenedesmaceae strains, Coelastrella astaxanthina Ki-4 and Scenedesmus obtusus Oki-4N; both strains were isolated under high light conditions. These AstaPs are classified as a novel family of carotenoprotein and are useful for providing valuable astaxanthin in water-soluble form; however, the distribution of AstaP orthologs in other microalgae remains unknown. Here, we examined the distribution of AstaP orthologs in the family Scenedesmaceae with two model microalgae, Chlamydomonas reinhardtii and Chlorella variabilis. The expression of AstaP orthologs under photooxidative stress conditions was detected in cell extracts of Scenedesmaceae strains, but not in model algal strains. Aqueous orange proteins produced by Scenedesmaceae strains were shown to bind astaxanthin. The protein from Scenedesmus costatus SAG 46.88 was purified. It was named ScosAstaP and found to bind astaxanthin. The deduced amino acid sequence from a gene encoding ScosAstaP showed 62% identity to Ki-4 AstaP. The expression of the genes encoding AstaP orthologs was shown to be inducible under photooxidative stress conditions; however, the production amounts of AstaP orthologs were estimated to be approximately 5 to 10 times lower than that of Ki-4 and Oki-4N.

Molecular identification of peptidoglycan recognition protein 5 and its functional characterization in innate immunity of large yellow croaker, Larimichthys crocea.

Fish peptidoglycan recognition proteins (PGRPs) play important roles in microbial recognition, and bacterial elimination. In the present study, a short-type PGRP from large yellow croaker, LcPGRP5 was cloned and its functions were characterized. LcPGRP5 gene encodes a protein containing conserved PGRP domain, but no signal peptide. Phylogenetic analysis shows that LcPGRP5 is clustered with other short PGRPs identified in other teleosts. LcPGRP5 is constitutively expressed in all tissues examined, with the highest expression being detected in the head kidney. Recombinant LcPGRP5 protein features amidase activity and bactericidal activity. Notably, LcPGRP5 could enhance the phagocytosis of the bacteria by large yellow croaker macrophage, with higher phagocytic capacity being observed in Staphylococcus aureus compared to Escherichia coli. Moreover, overexpression of LcPGRP5 suppresses pro-inflammatory effects elicited by bacterial exposure in the macrophage cell line. Overall, the present results clearly indicate the important roles of LcPGRP5 played in the innate immune responses against bacterial infection.

Validation of Recombinant Chicken Liver Bile Acid Binding Protein as a Tool for Cholic Acid Hosting.

Bile acids (BAs) are hydroxylated steroids derived from cholesterol that act at the intestinal level to facilitate the absorption of several nutrients and also play a role as signaling molecules. In the liver of various vertebrates, the trafficking of BAs is mediated by bile acid-binding proteins (L-BABPs). The ability to host hydrophobic or amphipathic molecules makes BABPs suitable for the distribution of a variety of physiological and exogenous substances. Thus, BABPs have been proposed as drug carriers, and more recently, they have also been employed to develop innovative nanotechnology and biotechnology systems. Here, we report an efficient protocol for the production, purification, and crystallization of chicken liver BABP (cL-BABP). By means of target expression as His6-tag cL-BABP, we obtained a large amount of pure and homogeneous proteins through a simple purification procedure relying on affinity chromatography. The recombinant cL-BABP showed a raised propensity to crystallize, allowing us to obtain its structure at high resolution and, in turn, assess the structural conservation of the recombinant cL-BABP with respect to the liver-extracted protein. The results support the use of recombinant cL-BABP for the development of drug carriers, nanotechnologies, and innovative synthetic photoswitch systems.

Structure of a consensus chitin-binding domain revealed by solution NMR.

Chitin-binding proteins (CBPs) are a versatile group of proteins found in almost every organism on earth. CBPs are involved in enzymatic carbohydrate degradation and also serve as templating scaffolds in the exoskeleton of crustaceans and insects. One specific chitin-binding motif found across a wide range of arthropods' exoskeletons is the "extended Rebers and Riddiford" consensus (R&R), whose mechanism of chitin binding remains unclear. Here, we report the 3D structure and molecular level interactions of a chitin-binding domain (CBD-γ) located in a CBP from the beak of the jumbo squid Dosidicus gigas. This CBP is one of four chitin-binding proteins identified in the beak mouthpart of D. gigas and is believed to interact with chitin to form a scaffold network that is infiltrated with a second set of structural proteins during beak maturation. We used solution state NMR spectroscopy to elucidate the molecular interactions between CBD-γ and the soluble chitin derivative pentaacetyl-chitopentaose (PCP), and find that folding of CBD-γ is triggered upon its interaction with PCP. To our knowledge, this is the first experimental 3D structure of a CBP containing the R&R consensus motif, which can be used as a template to understand in more details the role of the R&R motif found in a wide range of CBP-chitin complexes. The present structure also provides molecular information for biomimetic synthesis of graded biomaterials using aqueous-based chemistry and biopolymers.

Purification, secondary structure and antioxidant activity of metallothionein zinc-binding proteins from Arca subcrenata.

Zinc-binding proteins named MT-M-I and MT-M-II were obtained after purification from metal-exposed hairy clams (Arca subcrenata) using gel permeation and ion-exchange chromatography. MT-M-I and MT-M-II were resolved by ion-exchange chromatography, and they were found to have similar molecular weights. MT-M-I and MT-M-II can bind 6 and 7 equivalents of Zn2+ in vitro, and they showed unusual migration behaviors in Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (Tricine-SDS-PAGE). Such migration behaviors may be due to themetal thiolate clusters in these proteins. In terms of amino acid composition, the proportion of cysteine in MT-M-I and MT-M-II was approximately 30%, and glycine accounted for approximately 15%, where as aromatic amino acids were absent. Considering the performance in Tricine-SDS-PAGE and the amino acid compositions, MT-M-I and MT-M-II conform to the molecular characteristics of the metallothionein proteins. The structures were explored using circular dichroism (CD) and Fourier-transform infrared spectroscopy (FTIR). Also determined the antioxidant activities in terms of DPPH radical scavenging ability, hydroxyl radical (·OH) scavenging ability, and ferric-reducing/antioxidant power. The antioxidant activities of MT-M-I were found to be stronger than those of MT-M-II.

Structural Characterization of Act c 10.0101 and Pun g 1.0101-Allergens from the Non-Specific Lipid Transfer Protein Family.

(1) Background: Non-specific lipid transfer proteins (nsLTPs), which belong to the prolamin superfamily, are potent allergens. While the biological role of LTPs is still not well understood, it is known that these proteins bind lipids. Allergen nsLTPs are characterized by significant stability and resistance to digestion. (2) Methods: nsLTPs from gold kiwifruit (Act c 10.0101) and pomegranate (Pun g 1.0101) were isolated from their natural sources and structurally characterized using X-ray crystallography (3) Results: Both proteins crystallized and their crystal structures were determined. The proteins have a very similar overall fold with characteristic compact, mainly α-helical structures. The C-terminal sequence of Act c 10.0101 was updated based on our structural and mass spectrometry analysis. Information on proteins' sequences and structures was used to estimate the risk of cross-reactive reactions between Act c 10.0101 or Pun g 1.0101 and other allergens from this family of proteins. (4) Conclusions: Structural studies indicate a conformational flexibility of allergens from the nsLTP family and suggest that immunoglobulin E binding to some surface regions of these allergens may depend on ligand binding. Both Act c 10.0101 and Pun g 1.0101 are likely to be involved in cross-reactive reactions involving other proteins from the nsLTP family.

Tools and techniques for illuminating the cell biology of zinc.

Zinc (Zn2+) is an essential micronutrient that is required for a wide variety of cellular processes. Tools and methods have been instrumental in revealing the myriad roles of Zn2+ in cells. This review highlights recent developments fluorescent sensors to measure the labile Zn2+ pool, chelators to manipulate Zn2+ availability, and fluorescent tools and proteomics approaches for monitoring Zn2+-binding proteins in cells. Finally, we close with some highlights on the role of Zn2+ in regulating cell function and in cell signaling.

Isolation of Artificial Binding Proteins (Affimer Reagents) for Use in Molecular and Cellular Biology.

Artificial binding proteins have been validated as alternatives to antibodies in their use as research reagents in molecular and cellular biology. For example, they have been used as inhibitors of protein-protein interactions to modulate activity, to facilitate crystallization, and as probes for cellular imaging.Phage display is a widely used approach for isolating target-specific binding reagents, and it has even been used to isolate isoform-specific binding proteins and binders that can distinguish between highly homologous protein domains.Here, we describe methods that have been employed in isolating highly specific artificial binding proteins against a wide range of target proteins.

Engineering Af1521 improves ADP-ribose binding and identification of ADP-ribosylated proteins.

Protein ADP-ribosylation is a reversible post-translational modification that regulates important cellular functions. The identification of modified proteins has proven challenging and has mainly been achieved via enrichment methodologies. Random mutagenesis was used here to develop an engineered Af1521 ADP-ribose binding macro domain protein with 1000-fold increased affinity towards ADP-ribose. The crystal structure reveals that two point mutations K35E and Y145R form a salt bridge within the ADP-ribose binding domain. This forces the proximal ribose to rotate within the binding pocket and, as a consequence, improves engineered Af1521 ADPr-binding affinity. Its use in our proteomic ADP-ribosylome workflow increases the ADP-ribosylated protein identification rates and yields greater ADP-ribosylome coverage. Furthermore, generation of an engineered Af1521 Fc fusion protein confirms the improved detection of cellular ADP-ribosylation by immunoblot and immunofluorescence. Thus, this engineered isoform of Af1521 can also serve as a valuable tool for the analysis of cellular ADP-ribosylation under in vivo conditions.

Purified Sika deer antler protein attenuates GM-induced nephrotoxicity by activating Nrf2 pathway and inhibiting NF-κB pathway.

Although gentamicin is widely used as an antibiotic in clinical practice, it also has some side-effects, such as acute kidney injury, which is a common condition caused by the abuse of gentamicin. Sika deer antler protein (SDAPR) can antagonize drug-induced AKI. Since SDAPR is recognized as an effective part of velvet antler, its components were further separated. Two components named SDAP1 and SDAP2 were obtained. The protective effects of SDAPR, SDAP1 and SDAP2 on GM-induced cytotoxicity to HEK293 and its potential mechanisms were studied. MTT and xCELLigence Real-Time cell analysis showed that SDAPR, SDAP1 and SDAP2 could protect HEK293 cells from GM toxicity. Similarly, SDAPR, SDAP1 and SDAP2 can reduce ROS level, reduce oxidative stress and improve inflammation Further studies have shown that SDAPR, SDAP1 and SDAP2 upregulate the Nrf2/HO-1 pathway by increasing the expression of Nrf2 and HO-1, and down-regulate the NF-κB pathway by reducing the protein expression of NF-κB. Annexin V/PI flow cytometry and Hoechst 33258 staining showed that SDAPR, SDAP1 and SDAP2 inhibited GM-induced apoptosis in HEK293 cells. Western blot analysis showed SDAPR, SDAP1 and SDAP2 decreased expression level of Bax and Cleaved-caspase-3, and increased the expression level of Bcl-2. In addition, we examined the feasibility of SDAP1 and SDAP1 to avoid kidney injury in a GM mouse model. In conclusion, SDAPR, SDAP1 and SDAP2 can be used to prevent GM-induced HEK293 cytotoxicity, probably because they have strong anti-oxidative stress, anti-inflammatory and anti-apoptotic effects. And SDAP1 and SDAP2 can inhibit GM-induced acute kidney injury in mice.

Photooxidative stress-inducible orange and pink water-soluble astaxanthin-binding proteins in eukaryotic microalga.

Lipid astaxanthin, a potent antioxidant known as a natural sunscreen, accumulates in eukaryotic microalgae and confers photoprotection. We previously identified a photooxidative stress-inducible water-soluble astaxanthin-binding carotenoprotein (AstaP) in a eukaryotic microalga (Coelastrella astaxanthina Ki-4) isolated from an extreme environment. The distribution in eukaryotic microalgae remains unknown. Here we identified three novel AstaP orthologs in a eukaryotic microalga, Scenedesmus sp. Oki-4N. The purified proteins, named AstaP-orange2, AstaP-pink1, and AstaP-pink2, were identified as secreted fasciclin proteins with potent 1O2 quenching activity in aqueous solution, which are characteristics shared with Ki-4 AstaP. Nonetheless, the absence of glycosylation in the AstaP-pinks, the presence of a glycosylphosphatidylinositol (GPI) anchor motif in AstaP-orange2, and highly acidic isoelectric points (pI = 3.6-4.7), differed significantly from that of AstaP-orange1 (pI = 10.5). These results provide unique examples on the use of water-soluble forms of astaxanthin in photosynthetic organisms as novel strategies for protecting single cells against severe photooxidative stresses.

Novel Escherichia coli RNA Polymerase Binding Protein Encoded by Bacteriophage T5.

The Escherichia coli bacteriophage T5 has three temporal classes of genes (pre-early, early, and late). All three classes are transcribed by host RNA polymerase (RNAP) containing the σ70 promoter specificity subunit. Molecular mechanisms responsible for the switching of viral transcription from one class to another remain unknown. Here, we find the product of T5 gene 026 (gpT5.026) in RNAP preparations purified from T5-infected cells and demonstrate in vitro its tight binding to E. coli RNAP. While proteins homologous to gpT5.026 are encoded by all T5-related phages, no similarities to proteins with known functions can be detected. GpT5.026 binds to two regions of the RNAP ß subunit and moderately inhibits RNAP interaction with the discriminator region of σ70-dependent promoters. A T5 mutant with disrupted gene 026 is viable, but the host cell lysis phase is prolongated and fewer virus particles are produced. During the mutant phage infection, the number of early transcripts increases, whereas the number of late transcripts decreases. We propose that gpT5.026 is part of the regulatory cascade that orchestrates a switch from early to late bacteriophage T5 transcription.

The emm-Cluster Typing System.

emm-cluster typing system allows to classify most Streptococcus pyogenes variants into 48 different emm clusters. The system correlates nicely with the host serum binding capacities of the M proteins and has been used in epidemiological surveys, strain selection, and vaccine development. Here we describe the allocation of the emm cluster based on the emm-typing defining region.

Purification and Assays of Tachycitin.

An antimicrobial peptide tachycitin (73 amino acids) is purified by steps of chromatography, including Sephadex G-50 and S Sepharose FF, from the acid extract of hemocyte debris of horseshoe crabs. Tachycitin is present in monomer form in solution, revealed by ultracentrifugation analysis. Tachycitin exhibits bacterial agglutination activity and inhibits the growth of both Gram-negative bacteria, Gram-positive bacteria, and fungus Candida albicans. Interestingly, tachycitin shows synergistic antimicrobial activity in corporation with another antimicrobial peptide, big defensin. Tachycitin shows a specific binding activity to chitin but not to cellulose, mannan, xylan, and laminarin. Tachycitin is composed of the N-terminal three-stranded ß-sheet and the C-terminal two-stranded ß-sheet following a short helical turn, and the C-terminal structural motif shares a significant structural similarity with the chitin-binding domain derived from a plant chitin-binding protein, hevein.

FMNL2 regulates dynamics of fascin in filopodia.

Filopodia are peripheral F-actin-rich structures that enable cell sensing of the microenvironment. Fascin is an F-actin-bundling protein that plays a key role in stabilizing filopodia to support efficient adhesion and migration. Fascin is also highly up-regulated in human cancers, where it increases invasive cell behavior and correlates with poor patient prognosis. Previous studies have shown that fascin phosphorylation can regulate F-actin bundling, and that this modification can contribute to subcellular fascin localization and function. However, the factors that regulate fascin dynamics within filopodia remain poorly understood. In the current study, we used advanced live-cell imaging techniques and a fascin biosensor to demonstrate that fascin phosphorylation, localization, and binding to F-actin are highly dynamic and dependent on local cytoskeletal architecture in cells in both 2D and 3D environments. Fascin dynamics within filopodia are under the control of formins, and in particular FMNL2, that binds directly to dephosphorylated fascin. Our data provide new insight into control of fascin dynamics at the nanoscale and into the mechanisms governing rapid cytoskeletal adaptation to environmental changes. This filopodia-driven exploration stage may represent an essential regulatory step in the transition from static to migrating cancer cells.

Expression and purification of the heme exporter FLVCR1a.

With many crucial roles in enzymatic aerobic metabolism, the concentration of the heme must be tightly regulated. The heme exporter Feline Leukemia Virus sub-group C Receptor 1a (FLVCR1a), an integral membrane protein with twelve transmembrane helices, is a key player in the maintenance of cellular heme homeostasis. It was first identified as the host receptor for the Feline Leukemia Virus sub-group C (FeLV-C), a retrovirus causing hematological abnormalities in cats and other felines. Mutations in the Flvcr1 were later identified in human patients affected by Posterior Column Ataxia and Retinitis Pigmentosa (PCARP) and Hereditary Sensory and Autonomic Neuropathies (HSANs). Despite being an essential component in heme balance, currently there is a lack in the understanding of its function at the molecular level, including the effect of disease-causing mutations on protein function and structure. Therefore, there is a need for protocols to achieve efficient recombinant production yielding milligram amounts of highly pure protein to be used for biochemical and structural studies. Here, we report the first FLVCR1a reliable protocol suitable for both antibody generation and structural characterisation.

The structure of the TOM core complex in the mitochondrial outer membrane.

In the past three decades, significant advances have been made in providing the biochemical background of TOM (translocase of the outer mitochondrial membrane)-mediated protein translocation into mitochondria. In the light of recent cryoelectron microscopy-derived structures of TOM isolated from Neurospora crassa and Saccharomyces cerevisiae, the interpretation of biochemical and biophysical studies of TOM-mediated protein transport into mitochondria now rests on a solid basis. In this review, we compare the subnanometer structure of N. crassa TOM core complex with that of yeast. Both structures reveal remarkably well-conserved symmetrical dimers of 10 membrane protein subunits. The structural data also validate predictions of weakly stable regions in the transmembrane ß-barrel domains of the protein-conducting subunit Tom40, which signal the existence of ß-strands located in interfaces of protein-protein interactions.

Stabilization and Crystallization of a Membrane Protein Involved in Lipid Transport.

Lipoteichoic acids (LTA) are ubiquitous cell wall components of Gram-positive bacteria. In Staphylococcus aureus LTA are composed of a polymer with 1,3-linked glycerol phosphate repeating units anchored to the plasma membrane. The anchor molecule is a lipid-linked disaccharide (anchor-LLD) synthesized at the cytoplasmic leaflet of the membrane. The anchor lipid becomes accessible at the outer leaflet of the membrane after the flippase LtaA catalyzes translocation. Recently we have elucidated the structure of LtaA using vapor diffusion X-ray crystallography and in situ annealing. We were able to obtain LtaA crystals after optimization of purification protocols that led to stabilization of LtaA isolated in detergent micelles. Here we report a protocol that describes the purification, stabilization, crystallization, and data collection strategies carried out to determine the structure of LtaA. We highlight key points that can be used to determine crystal structures of other membrane proteins.

Ps19, a novel chitin binding protein from Pteria sterna capable to mineralize aragonite plates in vitro.

Mollusk shell is composed of two CaCO3 polymorphs (calcite and aragonite) and an organic matrix that consists of acetic acid- or ethylenediaminetetraacetic acid (EDTA)-soluble and insoluble proteins and other biomolecules (polysaccharides, ß-chitin). However, the shell matrix proteins involved in nacre formation are not fully known. Thus, the aim of this study was to identify and characterize a novel protein from the acetic acid-insoluble fraction from the shell of Pteria sterna, named in this study as Ps19, to have a better understanding of the biomineralization process. Ps19 biochemical characterization showed that it is a glycoprotein that exhibits calcium- and chitin-binding capabilities. Additionally, it is capable of inducing aragonite plate crystallization in vitro. Ps19 partial peptide sequence showed similarity with other known shell matrix proteins, but it displayed similarity with proteins from Crassostrea gigas, Mizuhopecten yessoensis, Biomphalaria glabrata, Alpysia californica, Lottia gigantea and Elysia chlorotica. The results obtained indicated that Ps19 might play an important role in nacre growth of mollusk shells.