Human lysozyme inhibits the in vitro aggregation of A beta peptides, which in vivo are associated with Alzheimer's disease

Biophysical Structural Chemistr

Expressing the human proteome for affinity proteomics: optimising expression of soluble protein domains and in vivo biotinylation

The generation of affinity reagents to large numbers of human proteins depends on the ability to express the target proteins as high-quality antigens. The Structural Genomics Consortium (SGC) focuses on the production and structure determination of human proteins. In a 7-year period, the SGC has deposited crystal structures of >800 human protein domains, and has additionally expressed and purified a similar number of protein domains that have not yet been crystallised. The targets include a diversity of protein domains, with an attempt to provide high coverage of protein families. The family approach provides an excellent basis for characterising the selectivity of affinity reagents. We present a summary of the approaches used to generate purified human proteins or protein domains, a test case demonstrating the ability to rapidly generate new proteins, and an optimisation study on the modification of >70 proteins by biotinylation in vivo. These results provide a unique synergy between large-scale structural projects and the recent efforts to produce a wide coverage of affinity reagents to the human proteome

Inhibiting and reversing amyloid-beta peptide (1-40) fibril formation with gramicidin S and engineered analogues

Biophysical Structural Chemistr

In Situ Proteolysis to Generate Crystals for Structure Determination: An Update

For every 100 purified proteins that enter crystallization trials, an average of 30 form crystals, and among these only 13–15 crystallize in a form that enables structure determination. In 2007, Dong et al reported that the addition of trace amounts of protease to crystallization trials—in situ proteolysis—significantly increased the number of proteins in a given set that produce diffraction quality crystals. 69 proteins that had previously resisted structure determination were subjected to crystallization with in situ proteolysis and ten crystallized in a form that led to structure determination (14.5% success rate). Here we apply in situ proteolysis to over 270 new soluble proteins that had failed in the past to produce crystals suitable for structure determination. These proteins had produced no crystals, crystals that diffracted poorly, or produced twinned and/or unmanageable diffraction data. The new set includes yeast and prokaryotic proteins, enzymes essential to protozoan parasites, and human proteins such as GTPases, chromatin remodeling proteins, and tyrosine kinases. 34 proteins yielded deposited crystal structures of 2.8 Å resolution or better, for an overall 12.6% success rate, and at least ten more yielded well-diffracting crystals presently in refinement. The success rate among proteins that had previously crystallized was double that of those that had never before yielded crystals. The overall success rate is similar to that observed in the smaller study, and appears to be higher than any other method reported to rescue stalled protein crystallography projects

A high-throughput immobilized bead screen for stable proteins and multi-protein complexes

We describe an in vitro colony screen to identify Escherichia coli expressing soluble proteins and stable, assembled multiprotein complexes. Proteins with an N-terminal 6His tag and C-terminal green fluorescent protein (GFP) S11 tag are fluorescently labeled in cells by complementation with a coexpressed GFP 1–10 fragment. After partial colony lysis, the fluorescent soluble proteins or complexes diffuse through a supporting filtration membrane and are captured on Talon® resin metal affinity beads immobilized in agarose. Images of the fluorescent colonies convey total expression and the level of fluorescence bound to the beads indicates how much protein is soluble. Both pieces of information can be used together when selecting clones. After the assay, colonies can be picked and propagated, eliminating the need to make replica plates. We used the method to screen a DNA fragment library of the human protein p85 and preferentially obtained clones expressing the full-length ‘breakpoint cluster region-homology' and NSH2 domains. The assay also distinguished clones expressing stable multi-protein complexes from those that are unstable due to missing subunits. Clones expressing stable, intact heterotrimeric E.coli YheNML complexes were readily identified in libraries dominated by complexes of YheML missing the N subunit

In Situ Proteolysis to Generate Crystals for Structure Determination: An Update

For every 100 purified proteins that enter crystallization trials, an average of 30 form crystals, and among these only 13–15 crystallize in a form that enables structure determination. In 2007, Dong et al reported that the addition of trace amounts of protease to crystallization trials—in situ proteolysis—significantly increased the number of proteins in a given set that produce diffraction quality crystals. 69 proteins that had previously resisted structure determination were subjected to crystallization with in situ proteolysis and ten crystallized in a form that led to structure determination (14.5% success rate). Here we apply in situ proteolysis to over 270 new soluble proteins that had failed in the past to produce crystals suitable for structure determination. These proteins had produced no crystals, crystals that diffracted poorly, or produced twinned and/or unmanageable diffraction data. The new set includes yeast and prokaryotic proteins, enzymes essential to protozoan parasites, and human proteins such as GTPases, chromatin remodeling proteins, and tyrosine kinases. 34 proteins yielded deposited crystal structures of 2.8 Å resolution or better, for an overall 12.6% success rate, and at least ten more yielded well-diffracting crystals presently in refinement. The success rate among proteins that had previously crystallized was double that of those that had never before yielded crystals. The overall success rate is similar to that observed in the smaller study, and appears to be higher than any other method reported to rescue stalled protein crystallography projects

SGC - Structural Biology and Human Health: A New Approach to Publishing Structural Biology Results

The Structural Genomics Consortium (SGC) is a not-for-profit, public-private partnership established to deliver novel structural biology knowledge on proteins of medical relevance and place this information into the public domain without restriction, spearheading the concept of "Open-Source Science" to enable drug discovery. The SGC is a major provider of structural information focussed on proteins related to human health, contributing 20.5% of novel structures released by the PDB in 2008. In this article we describe the PLoS ONE Collection entitled 'Structural Biology and Human Health: Medically Relevant Proteins from the SGC'. This Collection contains a series of articles documenting many of the novel protein structures determined by the SGC and work to further characterise their function. Each article in this Collection can be read in an enhanced version where we have integrated our interactive and intuitive 3D visualisation platform, known as iSee. This publishing platform enables the communication of complex structural biology and related data to a wide audience of non-structural biologists. With the use of iSee as the first example of an interactive and intuitive 3D document publication method as part of PLoS ONE, we are pushing the boundaries of structural biology data delivery and peer-review. Our strong desire is that this step forward will encourage others to consider the need for publication of three dimensional and associated data in a similar manner. © 2009 Lee et al

A Rapid Flp-In System for Expression of Secreted H5N1 Influenza Hemagglutinin Vaccine Immunogen in Mammalian Cells

Continuing transmissions of highly pathogenic H5N1 viruses in poultry and humans underscores the need for a rapid response to potential pandemic in the form of vaccine. Recombinant technologies for production of immunogenic hemagglutinin (HA) could provide an advantage over the traditional inactivated vaccine manufacturing process. Generation of stably transfected mammalian cells secreting properly folded HA proteins is important for scalable controlled manufacturing.We have developed a Flp-In based 293 stable cell lines through targeted site-specific recombination for expression of secreted hemagglutinin (HA) proteins and evaluated their immunogenicity. H5N1 globular domain HA1(1-330) and HA0(1-500) proteins were purified from the supernatants of 293 Flp-In stable cell lines. Both proteins were properly folded as confirmed by binding to H5N1-neutralizing conformation-dependent human monoclonal antibodies. The HA0 (with unmodified cleavage site) was monomeric, while the HA1 contained oligomeric forms. Upon rabbit immunization, both HA proteins elicited neutralizing antibodies against the homologous virus (A/Vietnam/1203/2004, clade 1) as well as cross-neutralizing antibodies against heterologous H5N1 clade 2 strains, including A/Indonesia/5/2005. These results exceeded the human antibody responses against the inactivated sub-virion H5N1 vaccine.Our data suggest that the 293 Flp-In system could serve as a platform for rapid expression of HA immunogens in mammalian cells from emerging influenza strains

Monalysin, a Novel ß-Pore-Forming Toxin from the Drosophila Pathogen Pseudomonas entomophila, Contributes to Host Intestinal Damage and Lethality

Pseudomonas entomophila is an entomopathogenic bacterium that infects and kills Drosophila. P. entomophila pathogenicity is linked to its ability to cause irreversible damages to the Drosophila gut, preventing epithelium renewal and repair. Here we report the identification of a novel pore-forming toxin (PFT), Monalysin, which contributes to the virulence of P. entomophila against Drosophila. Our data show that Monalysin requires N-terminal cleavage to become fully active, forms oligomers in vitro, and induces pore-formation in artificial lipid membranes. The prediction of the secondary structure of the membrane-spanning domain indicates that Monalysin is a PFT of the ß-type. The expression of Monalysin is regulated by both the GacS/GacA two-component system and the Pvf regulator, two signaling systems that control P. entomophila pathogenicity. In addition, AprA, a metallo-protease secreted by P. entomophila, can induce the rapid cleavage of pro-Monalysin into its active form. Reduced cell death is observed upon infection with a mutant deficient in Monalysin production showing that Monalysin plays a role in P. entomophila ability to induce intestinal cell damages, which is consistent with its activity as a PFT. Our study together with the well-established action of Bacillus thuringiensis Cry toxins suggests that production of PFTs is a common strategy of entomopathogens to disrupt insect gut homeostasis