mem-iLID, a fast and economic protein purification method.

Protein purification is the vital basis to study the function, structure and interaction of proteins. Widely used methods are affinity chromatography-based purifications, which require different chromatography columns and harsh conditions, such as acidic pH and/or adding imidazole or high salt concentration, to elute and collect the purified proteins. Here we established an easy and fast purification method for soluble proteins under mild conditions, based on the light-induced protein dimerization system improved light-induced dimer (iLID), which regulates protein binding and release with light. We utilize the biological membrane, which can be easily separated by centrifugation, as the port to anchor the target proteins. In Xenopus laevis oocyte and Escherichia coli, the blue light-sensitive part of iLID, AsLOV2-SsrA, was targeted to the plasma membrane by different membrane anchors. The other part of iLID, SspB, was fused with the protein of interest (POI) and expressed in the cytosol. The SspB-POI can be captured to the membrane fraction through light-induced binding to AsLOV2-SsrA and then released purely to fresh buffer in the dark after simple centrifugation and washing. This method, named mem-iLID, is very flexible in scale and economic. We demonstrate the quickly obtained yield of two pure and fully functional enzymes: a DNA polymerase and a light-activated adenylyl cyclase. Furthermore, we also designed a new SspB mutant for better dissociation and less interference with the POI, which could potentially facilitate other optogenetic manipulations of protein-protein interaction.

The M-phase regulatory phosphatase PP2A-B55δ opposes protein kinase A on Arpp19 to initiate meiotic division.

Oocytes are held in meiotic prophase for prolonged periods until hormonal signals trigger meiotic divisions. Key players of M-phase entry are the opposing Cdk1 kinase and PP2A-B55δ phosphatase. In Xenopus, the protein Arpp19, phosphorylated at serine 67 by Greatwall, plays an essential role in inhibiting PP2A-B55δ, promoting Cdk1 activation. Furthermore, Arpp19 has an earlier role in maintaining the prophase arrest through a second serine (S109) phosphorylated by PKA. Prophase release, induced by progesterone, relies on Arpp19 dephosphorylation at S109, owing to an unknown phosphatase. Here, we identified this phosphatase as PP2A-B55δ. In prophase, PKA and PP2A-B55δ are simultaneously active, suggesting the presence of other important targets for both enzymes. The drop in PKA activity induced by progesterone enables PP2A-B55δ to dephosphorylate S109, unlocking the prophase block. Hence, PP2A-B55δ acts critically on Arpp19 on two distinct sites, opposing PKA and Greatwall to orchestrate the prophase release and M-phase entry.

Highly synergistic antimicrobial activity of magainin 2 and PGLa peptides is rooted in the formation of supramolecular complexes with lipids.

Magainin 2 and PGLa are cationic, amphipathic antimicrobial peptides which when added as equimolar mixture exhibit a pronounced synergism in both their antibacterial and pore-forming activities. Here we show for the first time that the peptides assemble into defined supramolecular structures along the membrane interface. The resulting mesophases are quantitatively described by state-of-the art fluorescence self-quenching and correlation spectroscopies. Notably, the synergistic behavior of magainin 2 and PGLa correlates with the formation of hetero-domains and an order-of-magnitude increased membrane affinity of both peptides. Enhanced membrane association of the peptide mixture is only observed in the presence of phophatidylethanolamines but not of phosphatidylcholines, lipids that dominate bacterial and eukaryotic membranes, respectively. Thereby the increased membrane-affinity of the peptide mixtures not only explains their synergistic antimicrobial activity, but at the same time provides a new concept to increase the therapeutic window of combinatorial drugs.

Histone chaperone exploits intrinsic disorder to switch acetylation specificity.

Histones, the principal protein components of chromatin, contain long disordered sequences, which are extensively post-translationally modified. Although histone chaperones are known to control both the activity and specificity of histone-modifying enzymes, the mechanisms promoting modification of highly disordered substrates, such as lysine-acetylation within the N-terminal tail of histone H3, are not understood. Here, to understand how histone chaperones Asf1 and Vps75 together promote H3 K9-acetylation, we establish the solution structural model of the acetyltransferase Rtt109 in complex with Asf1 and Vps75 and the histone dimer H3:H4. We show that Vps75 promotes K9-acetylation by engaging the H3 N-terminal tail in fuzzy electrostatic interactions with its disordered C-terminal domain, thereby confining the H3 tail to a wide central cavity faced by the Rtt109 active site. These fuzzy interactions between disordered domains achieve localization of lysine residues in the H3 tail to the catalytic site with minimal loss of entropy, and may represent a common mechanism of enzymatic reactions involving highly disordered substrates.

Microtubule end conversion mediated by motors and diffusing proteins with no intrinsic microtubule end-binding activity.

Accurate chromosome segregation relies on microtubule end conversion, the ill-understood ability of kinetochores to transit from lateral microtubule attachment to durable association with dynamic microtubule plus-ends. The molecular requirements for this conversion and the underlying biophysical mechanisms are elusive. We reconstituted end conversion in vitro using two kinetochore components: the plus end-directed kinesin CENP-E and microtubule-binding Ndc80 complex, combined on the surface of a microbead. The primary role of CENP-E is to ensure close proximity between Ndc80 complexes and the microtubule plus-end, whereas Ndc80 complexes provide lasting microtubule association by diffusing on the microtubule wall near its tip. Together, these proteins mediate robust plus-end coupling during several rounds of microtubule dynamics, in the absence of any specialized tip-binding or regulatory proteins. Using a Brownian dynamics model, we show that end conversion is an emergent property of multimolecular ensembles of microtubule wall-binding proteins with finely tuned force-dependent motility characteristics.

Expression, purification and crystallization of the novel Xenopus tropicalis ALDH16B1, a homologue of human ALDH16A1.

ALDH16 is a novel family of the aldehyde dehydrogenase (ALDH) superfamily with unique structural characteristics that distinguish it from the other ALDH superfamily members. In addition to structural characteristics, there is an evolutionary-related grouping within the ALDH 16 genes. The ALDH16 isozymes in frog, lower animals, and bacteria possess a critical Cys residue in their active site, which is absent from ALDH16 in mammals and fish. Genomic analysis and plasma metabolomic studies have associated ALDH16A1 with the pathogenesis of gout in humans, although its actual involvement in this disease is poorly understood. Insight into the structure of ALDH16A1 is an important step in deciphering its function in gout. Herein, we report our efforts towards the structural characterization of Xenopus tropicalis ALDH16B1 (the homolog of human ALDH16A1) that was predicted to be catalytically-active. Recombinant ALDH16B1 was expressed in Sf9 cells and purified using affinity and size exclusion chromatography. Crystallization of ALDH16B1 was achieved by vapor diffusion. A data set was collected at 2.5 Šand preliminary crystallographic analysis showed that the frog ALDH16B1 crystals belong to the P 212 121 space group with unit cell parameters a = 80.48 Å, b = 89.73 Å, c = 190.92 Å, α = ß = γ = 90.00°. Structure determination is currently in progress.

Protein Immunodepletion and Complementation in Xenopus laevis Egg Extracts.

The Xenopus egg extract system has been widely used to study cell cycle events, including DNA replication, nuclear envelope formation, spindle assembly, chromosome condensation and kinetochore formation. The functional roles of the proteins involved in these processes can be determined by immunodepleting a protein of interest from the extract. As immunodepletion may result in co-depletion of other proteins, the protein of interest can be added back to the extract to verify its function. Additionally, proteins harboring point mutations or domain deletions may be added to assess their functions. Here we outline the immunodepletion procedure and two separate methods for restoring a protein of interest: addition of either a recombinant protein or an mRNA that supports translation in egg extracts. The tradeoffs between these two methods are discussed.

Transition metal dependent regulation of the signal transduction cascade driving oocyte meiosis.

The G2-M transition of the cell cycle requires the activation of members of the Cdc25 dual specificity phosphatase family. Using Xenopus oocyte maturation as a model system, we have previously shown that chelation of transition metals blocks meiosis progression by inhibiting Cdc25C activation. Here, using approaches that allow for the isolation of very pure and active recombinant Cdc25C, we show that Cdc25C does not bind zinc as previously reported. Additionally, we show that mutants in the disordered C-terminal end of Cdc25C are poor initiators of meiosis, likely due to their inability to localize to the proper sub-cellular location. We further demonstrate that the transition metal chelator, TPEN, acts on or upstream of polo-like kinases in the oocyte to block meiosis progression. Together our results provide novel insights into Cdc25C structure-function relationship and the role of transition metals in regulating meiosis.

Expression and purification of histone H3 proteins containing multiple sites of lysine acetylation using nonsense suppression.

Lysine acetylation is a common post-translational modification, which is especially prevalent in histone proteins in chromatin. A number of strategies exist for generating histone proteins containing lysine acetylation, but an especially attractive approach is to genetically encode acetyl-lysine residues using nonsense suppression. This strategy has been successfully applied to single sites of histone acetylation. However, because histone acetylation can often occur at multiple sites simultaneously, we were interested in determining whether this approach could be extended. Here we show that we can express histone H3 proteins that incorporate up to four sites of lysine acetylation on the histone tail. Because the amount of expressed multi-acetylated histone is reduced relative to the wild type, a purification strategy involving affinity purification and ion exchange chromatography was optimized. This expression and purification strategy ultimately generates H3 histone uniformly acetylated at the desired position at levels and purity sufficient to assemble histone octamers. Histone octamers containing four sites of lysine acetylation were assembled into mononucleosomes and enzymatic assays confirmed that this acetylation largely blocks further acetylation by the yeast SAGA acetyltransferase complex.

A novel approach to the expression and purification of recombinant Xenopus Cdc25C.

The Cdc25 family encodes dual specificity protein phosphatases that play critical roles in cell cycle progression. Activation of the Cdc25C represents a primary driver for meiosis progression in Xenopus oocytes. Given its central role in meiosis the Xenopus Cdc25C has been studied extensively, however purification of the recombinant protein is difficult thus preventing better characterization of its function. Here we describe methods to overcome these difficulties resulting in the production of high purity and yield recombinant Xenopus Cdc25C. We use a synthetic Xenopus Cdc25C gene that was codon optimized for expression in E. coli. We further combine an N-terminal His-tag with a C-terminal Strep-tag II, to isolate extremely pure full-length Cdc25C protein. The recombinant Xenopus Cdc25C is active both in vitro using a phosphatase assay and in vivo when injected into Xenopus oocytes. This new approach should be applicable to the purification of other members of the Cdc25 gene family.

Identification of novel peptides from amphibian (Xenopus tropicalis) skin by direct tissue MALDI-MS analysis.

Twelve novel peptides (Pxt-1 to Pxt-12) were isolated from the skin of Xenopus tropicalis, diploid frogs, using topological MS analysis. Among them, Pxt-8, Pxt-9, and Pxt-10 were the N terminus of Pxt-1, N terminus of Pxt-3 and C terminus of Pxt-11, respectively. The Pxt-3 and Pxt-11 peptides shared significant sequence homologies with magainins 1, -2 and levitide, respectively, which all isolated from X. laevis. Pxt-12 was identical to the X. tropicalis XT-6-like precursor previously isolated by ESI-MS/MS. None of the Pxt peptides contained any Cys, Asp, Tyr or Trp, although Leu and Lys were frequently found as typical frog-skin peptides. RT-PCR analysis confirmed the gene expressions of Pxt-2, Pxt-3, Pxt-4, Pxt-5, Pxt-7 and Pxt-11 in X. tropicalis skin. Several ion peaks corresponding to all identified Pxt peptides were observed with MALDI-MS analysis of X. tropicalis secretory fluids, collected after in vivo stimulation, which suggested that Pxt peptides were definitely secretory molecules. CD studies and Schiffer-Edmundson helical wheel projections suggested that Pxt-5, as well as mastoparan, at least, could form a typical amphiphilic α helix without a phospholipid or a membrane-mimetic solvent (trifluoroethanol). Moreover, Pxt-2 and Pxt-5 showed growth inhibitory effects on both Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive). Measurements of dynamic light scattering and the surface tensions of Pxt peptides solutions suggested that both Pxt-2 and Pxt-5 could form associations as micelles and behave like a general surfactant. Moreover, the remarkable foaming properties of Pxt-2 and Pxt-5 were observed, as well as those of the secretory fluids of X. tropicalis.

Evidence from peptidomic analysis of skin secretions that allopatric populations of Xenopus gilli (Anura:Pipidae) constitute distinct lineages.

The International Union for Conservation of Nature (IUCN) Endangered Cape Platanna Xenopus gilli inhabits disjunct ranges at the tip of Cape Peninsula and near the town of Kleinmond on opposite sides of False Bay in the extreme southwest of Africa. Peptidomic analysis of host-defense peptides in norepinephrine-stimulated skin secretions from frogs from the Cape Peninsula range resulted in the identification of two magainins, two peptide glycine-leucine-amide (PGLa) peptides, two xenopsin-precursor fragment (XPF) peptides, nine caerulein-precursor fragment (CPF) peptides, and a peptide related to peptide glycine-glutamine (PGQ) previously found in an extract of Xenopus laevis stomach. The primary structures of the peptides indicate a close phylogenetic relationship between X. gilli and X. laevis but only magainin-1, PGLa and one CPF peptide are identical in both species. Consistent with previous data, the CPF peptides show the greatest antimicrobial potency but are hemolytic. There are appreciable differences in the expression of host-defense peptide genes in frogs from the population of animals sampled near Kleinmond as peptides corresponding to magainin-G2, XPF-G1, XPF-G2, and four CPF peptides, present in secretions from the Cape Peninsula frogs, were not identified in the skin secretions from Kleinmond frogs. Conversely, PGLa-G3, XPF-G3, and three CPF peptides were identified in the Kleinmond frogs but not in the Cape Peninsula animals. The data support the conclusion from morphometric analyses and comparisons of the nucleotide sequences of mitochondrial genes that the disjunct populations of X. gilli have undergone appreciable genetic, morphological, and phenotypic divergence.

Antimicrobial and immunomodulatory properties of PGLa-AM1, CPF-AM1, and magainin-AM1: potent activity against oral pathogens.

Cationic amphipathic α-helical peptides are intensively studied classes of host defence peptides (HDPs). Three peptides, peptide glycine-leucine-amide (PGLa-AM1), caerulein-precursor fragment (CPF-AM1) and magainin-AM1, originally isolated from norepinephrine-stimulated skin secretions of the African volcano frog Xenopus amieti (Pipidae), were studied for their antimicrobial and immunomodulatory activities against oral and respiratory pathogens. Minimal effective concentrations (MECs), determined by radial diffusion assay, were generally lower than minimal inhibitory concentrations (MICs) determined by microbroth dilution. PGLa-AM1 and CPF-AM1 were particularly active against Streptococcus mutans and all three peptides were effective against Fusobacterium nucleatum, whereas Enterococcus faecalis and Candida albicans proved to be relatively resistant micro-organisms. A type strain of Pseudomonas aeruginosa was shown to be more susceptible than the clinical isolate studied. PGLa-AM1 displayed the greatest propensity to bind lipopolysaccharide (LPS) from Escherichia coli, P. aeruginosa and Porphyromonas gingivalis. All three peptides showed less binding to P. gingivalis LPS than to LPS from the other species studied. Oral fibroblast viability was unaffected by 50 µM peptide treatments. Production of the pro-inflammatory cytokine IL-8 by oral fibroblasts was significantly increased following treatment with 1 or 10 µM magainin-AM1 but not following treatment with PGLa-AM1 or CPF-AM1. In conclusion, as well as possessing potent antimicrobial actions, the X. amieti peptides bound to LPS from three human pathogens and had no effect on oral fibroblast viability. CPF-AM1 and PGLa-AM1 show promise as templates for the design of novel analogues for the treatment of oral and dental diseases associated with bacteria or fungi.

Sperm and spermatids contain different proteins and bind distinct egg factors.

Spermatozoa are more efficient at supporting normal embryonic development than spermatids, their immature, immediate precursors. This suggests that the sperm acquires the ability to support embryonic development during spermiogenesis (spermatid to sperm maturation). Here, using Xenopus laevis as a model organism, we performed 2-D Fluorescence Difference Gel Electrophoresis (2D-DIGE) and mass spectrometry analysis of differentially expressed proteins between sperm and spermatids in order to identify factors that could be responsible for the efficiency of the sperm to support embryonic development. Furthermore, benefiting from the availability of egg extracts in Xenopus, we also tested whether the chromatin of sperm could attract different egg factors compared to the chromatin of spermatids. Our analysis identified: (1) several proteins which were present exclusively in sperm; but not in spermatid nuclei and (2) numerous egg proteins binding to the sperm (but not to the spermatid chromatin) after incubation in egg extracts. Amongst these factors we identified many chromatin-associated proteins and transcriptional repressors. Presence of transcriptional repressors binding specifically to sperm chromatin could suggest its preparation for the early embryonic cell cycles, during which no transcription is observed and suggests that sperm chromatin has a unique protein composition, which facilitates the recruitment of egg chromatin remodelling factors. It is therefore likely that the acquisition of these sperm-specific factors during spermiogenesis makes the sperm chromatin suitable to interact with the maternal factors and, as a consequence, to support efficient embryonic development.

Antimicrobial activities and membrane-active mechanism of CPF-C1 against multidrug-resistant bacteria, a novel antimicrobial peptide derived from skin secretions of the tetraploid frog Xenopus clivii.

Hospital-acquired infections caused by multidrug-resistant bacteria pose significant challenges for treatment, which necessitate the development of new antibiotics. Antimicrobial peptides are considered potential alternatives to conventional antibiotics. The skin of Anurans (frogs and toads) amphibians is an extraordinarily rich source of antimicrobial peptides. CPF-C1 is a typical cationic antimicrobial peptide that was originally isolated from the tetraploid frog Xenopus clivii. Our results showed that CPF-C1 has potent antimicrobial activity against both sensitive and multidrug-resistant bacteria. It disrupted the outer and inner membranes of bacterial cells. CPF-C1 induced both propidium iodide uptake into the bacterial cell and the leakage of calcein from large liposome vesicles, which suggests a mode of action that involves membrane disturbance. Scanning electron microscopy and transmission electron microscopy verified the morphologic changes of CPF-C1-treated bacterial cells and large liposome vesicles. The membrane-dependent mode of action signifies that the CPF-C1 peptide functions freely and without regard to conventional resistant mechanisms. Additionally, it is difficult for bacteria to develop resistance against CPF-C1 under this action mode. Other studies indicated that CPF-C1 had low cytotoxicity against mammalian cell. In conclusion, considering the increase in multidrug-resistant bacterial infections, CPF-C1 may offer a new strategy that can be considered a potential therapeutic agent for the treatment of diseases caused by multidrug-resistant bacteria.

[The obtaining and analysis of physiological activity of secreted proteins of Noggin family].

We have developed methods for producing recombinant proteins of Noggin family (Noggin1 and Noggin2 of the Xenopus laevis frog) that can interact with BMP factors of TGF-beta superfamily. The genetic constructs which allow one to effectively obtain Noggin1 and Noggin2 from synthetic mRNA microinjected into Xenopus laevis early embryos, as well as in the prokaryotic expression system, were generated. The obtained proteins contain three Myc-tag epitopes on their N-terminus. This allow one to compare the expression levels of Noggin1 and Noggin 2 constructs, to purify them on the affine immunosorbent and to show the activity of Noggin proteins by analyzing their ability to bind BMP4 factor TGF-beta surperfamily by co-immunoprecipitation.

Bacterial lipopolysaccharides stimulate production of XCL1, a calcium-dependent lipopolysaccharide-binding serum lectin, in Xenopus laevis.

Xenopus laevis serum lectin XCL1 is a newly identified molecule of the XCGL (or X-lectin) family, a unique group of Ca(2+)-dependent lectins that have a fibrinogen-like domain. The XCL1 protein was purified from lipopolysaccharide (LPS)-stimulated frog sera by sequential affinity chromatography on heparin-acrylic beads and galactose-Sepharose. XCL1 comprises multiple oligomeric proteins consisting of 37-kDa subunit polypeptides, as revealed by sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) and Western blot analyses using the monoclonal antibody (mAb) produced against the recombinant XCL1 polypeptide. In the presence of Ca(2+), the protein bound to Escherichia coli, Staphylococcus aureus, LPS and galactose and the bound XCL1 was competitively eluted using ribose and xylose, and the elution was as efficient as that using EDTA, whereas elution using hexoses, GalNAc or GlcNAc was less effective. In reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses, XCL1 expression was ubiquitously detected in frog tissues, with relatively high levels in hematopoietic tissues including the spleen, liver and kidney. Intraperitoneal injection of E. coli, S. aureus or 100-300µg S-type LPS from various bacteria induced several-fold increases in serum XCL1 concentrations on day 3, and the elevated levels retained up to day 12. It also caused a remarkable increase of the splenic XCL1 expression on day 3, followed by a rapid decline to nearly nonstimulated control levels by day 7. The R-type LPS with shortened polysaccharide chains was less effective in inducing the serum XCL1 response, indicating that the sugar chains of LPS were important, if not essential, for the stimulation of XCL1 production. These results suggest that XCL1 is a pathogen recognition molecule involved in antimicrobial innate immunity in Xenopus.

Recombinant expression and purification of the antimicrobial peptide magainin-2.

Magainin-2 (MAG2) is a polycationic antimicrobial peptide isolated from the skin of the African clawed frog Xenopus laevis. It has a wide spectrum of antimicrobial activities against gram-positive and gram-negative bacteria, fungi, and induces osmotic lysis of protozoa. MAG2 also possesses antiviral and antitumoral properties. These activities make this peptide a promising candidate for therapeutic applications. Recombinant expression systems are necessary for the affordable production of large amounts of the biologically active peptide. In this work, MAG2 has been cloned to the N-terminal of a family III carbohydrate-binding module fused to the linker sequence (LK-CBM3) from Clostridium thermocellum; a formic acid recognition site was introduced between the two modules for chemical cleavage of the peptide. The recombinant protein MAG2-LK-CBM3 was expressed in Escherichia coli BL21 (DE3) and MAG2 was successfully cleaved and purified from the fusion partner LK-CBM3. Its functionality was confirmed by testing its activity against gram-negative bacteria.

Caerulein precursor fragment (CPF) peptides from the skin secretions of Xenopus laevis and Silurana epitropicalis are potent insulin-releasing agents.

Peptidomic analysis of norepinephrine-stimulated skin secretions of the tetraploid clawed frog Xenopus laevis (Pipidae) led to the identification of 10 peptides with the ability to stimulate the release of insulin from the rat BRIN-BD11 clonal ß cell line. These peptides were purified to near homogeneity and structural characterization showed that they belong to the magainin (2 peptides), peptide glycine-leucine-amide (PGLa) (1 peptide), xenopsin precursor fragment (1 peptide), and caerulein precursor fragment (CPF) (6 peptides) families. CPF-1, CPF-3, CPF-5 and CPF-6 were the most potent producing a significant (P < 0.05) increase in the rate of insulin release at concentration of 0.03 nM. CPF-7 (GFGSFLGKALKAALKIGANALGGAPQQ) produced the maximum stimulation of insulin release (571 ± 30% of basal rate at 3 µM). In addition, CPF-SE1 (GFLGPLLKLGLKGVAKVIPHLIPSRQQ), previously isolated from skin secretions of the tetraploid frog Silurana epitropicalis, produced a significant (P < 0.05) increase in the rate of insulin release at 0.03 nM with a 514 ± 13% increase over basal rate at 3 µM. No CPF peptide stimulated release of the cytosolic enzyme, lactate dehydrogenase from BRIN-BD11 cells at concentrations up to 3 µM indicating that the integrity of the plasma membrane had been preserved. The mechanism of action of the CPF peptides involves, at least in part, membrane depolarization and an increase in intracellular Ca(2+) concentration. The CPF peptides show potential for development into agents for the treatment of Type 2 diabetes.

Immunoisolation of protein complexes from Xenopus.

The immunoaffinity isolation of protein complexes is an essential technique for the purification and -concentration of protein complexes from cells and tissues. In this chapter we present the methodologies for the purification of proteins and protein complexes from Xenopus laevis and Xenopus tropicalis. Specific to this protocol are the techniques for the cryolysis of Xenopus cells and tissues, a procedure that limits contamination from yolk proteins while preserving endogenous protein complexes, the methodologies for immunoaffinity purification of proteins using magnetic beads, and the protocols for western blot analysis. In addition, the procedures in this chapter can be extended to use with proteomic analysis of protein complexes as presented in the following chapter.