Protein Libraries Research Articles

An overview of yeast cell wall proteins and their contribution in yeast display system

Yeast surface display has become an increasingly popular tool for protein engineering and library screening applications. Although, recent advances have greatly expanded the capability of yeast surface display, the protein display system is still far away from industrial application. One of the major components of a stable, efficient and successful yeast surface display system is cell wall anchor protein with which our desired foreign protein will be attached. We studied 80 different yeast cell wall anchored proteins originated mostly from Saccharomyces cerevisiae and Candida albicans. We studied in details all the cell wall proteins in order to find out suitable cell wall proteins to recommend for the researchers to use in the construction of yeast display system. We considered selective physical properties of different yeast cell wall proteins that are crucial for selecting best suited cell surface anchor proteins which are molecular weight, binding domain of anchor protein, length of amino acid and fusion site. Finally, our studies showed that Ccw11, Ccw12. Cwp1, Cwp2, Dan1, Gas1, Gas5, Exg1, Ycr89, Ecm33, Pga4, Sap9, Sap10, Pst1, Pir1, Pir2, Pir3, Pir4, Cis1, Scw4, Scw6, Bgl2, Uth1, Scw1 are the promising and suitable cell wall anchor proteins could be used in construction of yeast cell surface display system. Additionally, this review presents detailed information about all the cell wall proteins in a single work. The future researchers in this field will be able to construct more efficient yeast display system for recombinant protein production at industrial scale using the knowledge presented in this work.
 Asian J. Med. Biol. Res. June 2019, 5(4): 246-257

Single-molecule Protein Sequencing using Biological Nanopores

The next wave of research in bioinformatics and fundamental biology will revolve around sequencing the enormous and dynamic variations in the proteome. A method for sequencing proteins and detecting PTMs at the single-molecule level would be revolutionary for proteomics research. We report proof-of-concept and initial results for a new single-molecule protein sequencing technique. We use a typical enzyme-controlled biological nanopore sequencing setup, with the addition of a peptide fragment covalently linked to the end of the DNA strand. The nanopore sequencer first reads the DNA, then the peptide fragment as the DNA-peptide conjugate is pulled up through the nanopore. The readout is then used to decode the peptide sequence, map substitutions and post-translation modifications, or identify the peptide from a library of proteins. Similarly to nanopore DNA sequencing, this technique is in principle capable of single-molecule measurements, high throughput, low experimental overhead and minimal sample preparation, and sensitivity to changes in individual monomers.

Measuring the Physical Properties of Synthetic Cement Derived Barnacle Adhesive Nanomaterials from the Barnacle Amphibalanus amphitrite

Barnacles, a common biofouling organism, produce a micron-thick layer of long-lasting, proteinaceous, amyloid-like underwater adhesive. The barnacle triggers assembly and delivery of glue materials from a liquid, which set as a bulk adhesive gel in seawater environments. To accomplish this, barnacles use an organized co-block arrangement of sequence domains to dictate material formation, cohesive strength, and adhesive bonding properties of larger protein components. To understand physical properties of barnacle glue and construct a new class of underwater adhesive biomaterials, we have established a materials library from barnacle protein sequences for study. Derived from consensus barnacle cement sequences of Amphibalanus amphitrite natural glue proteins, we use eight synthetic barnacle cement peptides (BCPs), with patterned sequence motifs from the natural adhesive, for materials processing and testing. To understand how barnacle adhesive is fabricated, we looked closely at the BCP sequence to evaluate its function in delivery, polymerization, curing, and adhesion. Bulk gel materials, characterized by atomic force microscopy and profilometry, are shown to form dependent upon a BCP sequence pattern. The adhesive properties of assembled BCP gels were measured using adhesion testing of pull off (normal force) and push off (shear force). In shear force, patterned sequences behave differently demonstrating the structuring of fibrils enhances resistance to shear. Bulk BCP gels are shown to be pressure sensitive and have similar strength to natural barnacle glue. The alternating patterns identified suggest glue chemistries are displayed in the BCPs. Using BCPs, we link distinct peptide sequences to polymerization, recognition, formation of fibrils, and the physical properties of bulk gel materials. We offer a means to develop and test a protein library to establish the relationship between dry adhesion and biomaterial structure for insights into how natural barnacles adhere.

A high-throughput screening method to identify proteins involved in unfolded protein response of the endoplasmic reticulum in plants

BackgroundThe unfolded protein response (UPR) is a highly conserved process in eukaryotic organisms that plays a crucial role in adaptation and development. While the most ubiquitous components of this pathway have been characterized, current efforts are focused on identifying and characterizing other UPR factors that play a role in specific conditions, such as developmental changes, abiotic cues, and biotic interactions. Considering the central role of protein secretion in plant pathogen interactions, there has also been a recent focus on understanding how pathogens manipulate their host’s UPR to facilitate infection.ResultsWe developed a high-throughput screening assay to identify proteins that interfere with UPR signaling in planta. A set of 35 genes from a library of secreted proteins from the maize pathogen Ustilago maydis were transiently co-expressed with a reporter construct that upregulates enhanced yellow fluorescent protein (eYFP) expression upon UPR stress in Nicotiana benthamiana plants. After UPR stress induction, leaf discs were placed in 96 well plates and eYFP expression was measured. This allowed us to identify a previously undescribed fungal protein that inhibits plant UPR signaling, which was then confirmed using the classical but more laborious qRT-PCR method.ConclusionsWe have established a rapid and reliable fluorescence-based method to identify heterologously expressed proteins involved in UPR stress in plants. This system can be used for initial screens with libraries of proteins and potentially other molecules to identify candidates for further validation and characterization.

Enrichment of rare events using a multi-parameter high throughput microfluidic droplet sorter.

High information content analysis, enrichment, and selection of rare events from a large population are of great importance in biological and biomedical research. The fluorescence lifetime of a fluorophore, a photophysical property which is independent of and complementary to fluorescence intensity, has been incorporated into various imaging and sensing techniques through microscopy, flow cytometry and droplet microfluidics. However, the throughput of fluorescence lifetime activated droplet sorting is orders of magnitude lower than that of fluorescence activated cell sorting, making it unattractive for applications such as directed evolution of enzymes, despite its highly effective compartmentalization of library members. We developed a microfluidic sorter capable of selecting fluorophores based on fluorescence lifetime and brightness at two excitation and emission colors at a maximum droplet rate of 2.5 kHz. We also present a novel selection strategy for efficiently analyzing and/or enriching rare fluorescent members from a large population which capitalizes on the Poisson distribution of analyte encapsulation into droplets. The effectiveness of the droplet sorter and the new selection strategy are demonstrated by enriching rare populations from a ∼108-member site-directed mutagenesis library of fluorescent proteins expressed in bacteria. This selection strategy can in principle be employed on many droplet sorting platforms, and thus can potentially impact broad areas of science where analysis and enrichment of rare events is needed.

In silico design of two novel fusion proteins, p28-IL-24 and p28-M4, targeted to breast cancer cells.

Background and purpose:An anticancer peptide P28, has shown to be cytolethal on various cancer cells including breast cancer. Moreover, p28 can be also used as a targeting moiety in the structure of fusion proteins. IL-24 (or its truncated form, M4) is a cytokine with anticancer activity against a wide range of tumor cells. We aimed at production of a fusion protein consisted of p28 and either IL-24 or M4 to target breast cancer. However, selection of a proper linker to join the two moieties without intervening each other’s function is a key factor in the construction of fusion proteins. In the present study, the impact of different linkers on construction of the two chimeric proteins (p28-IL-24 and p28-M4) was assessed in silico.Experimental approach:After selection of some linkers with different lengths and characteristics, a small library of the chimeric proteins was created and assessed. Furthermore, following selection of the most suitable linker, the three-dimensional structures and dynamic behavior of both fusion proteins were evaluated by homology modeling and molecular dynamic simulation, respectively.Findings / Results:Based on the results, a rigid linker having the peptide sequences of AEAAAKEAAAKA showed highest freedom of action for both moieties.Conclusion and implications:Between the p28-IL-24 and p28-M4 fusion proteins, the former showed better stability as well as solubility and might show stronger anticancer effects in vitro and in vivo, because its peptide moieties showed to exert their activities freely.

A convenient protein library for spectroscopic calibrations

While several Raman, CD or FTIR spectral libraries are available for well-characterized proteins of known structure, proteins themselves are usually very difficult to acquire, preventing a convenient calibration of new instruments and new recording methods. The problem is particularly critical in the field of FTIR spectroscopy where numerous new methods are becoming available on the market.The present papers reports the construction of a protein library (cSP92) including commercially available products, that are well characterized experimentally for their purity and solubility in conditions compatible with the recording of FTIR spectra and whose high-resolution structure is available.Overall, 92 proteins were selected. These proteins cover well the CATH space at the level of classes and architectures. In terms of secondary structure content, an analysis of their high-resolution structure by DSSP shows that the mean content in the different secondary structures present in cSP92 is very similar to the mean content found in the PDB.The 92-protein set is analyzed in details for the distribution of helix length, number of strands in β- sheets, length of β-strands and amino acid content, all features that may be important for the interpretation of FTIR spectra.

An efficient single pot DNA recombination method for protein library generation

A simple single pot method was developed to generate a library of protein hybrids/chimeras from unrelated parents. This method allows unbiased facile DNA shuffling based recombination between the parents with the introduction of type IIB restriction enzyme, BsaXI, at multiple distinct crossover sites. To assess this methodology, B1 immunoglobulin domain of protein G and top7 were recombined at four sites, yielding 25 (32) possible hybrid proteins. Tandem colony multiplex PCR based screening was followed to screen the hybrid clones and, sequencing analysis confirmed seven hybrid clones with no sequence bias.

On the possible origin of protein homochirality, structure, and biochemical function

Living systems have chiral molecules, e.g., native proteins that almost entirely contain L-amino acids. How protein homochirality emerged from a background of equal numbers of L and D amino acids is among many questions about life's origin. The origin of homochirality and its implications are explored in computer simulations examining the stability and structural and functional properties of an artificial library of compact proteins containing 1:1 (termed demi-chiral), 3:1, and 1:3 ratios of D:L and purely L or D amino acids generated without functional selection. Demi-chiral proteins have shorter secondary structures and fewer internal hydrogen bonds and are less stable than homochiral proteins. Selection for hydrogen bonding yields a preponderance of L or D amino acids. Demi-chiral proteins have native global folds, including similarity to early ribosomal proteins, similar small molecule ligand binding pocket geometries, and many constellations of L-chiral amino acids with a 1.0-Å RMSD to native enzyme active sites. For a representative subset containing 550 active site geometries matching 457 (2) 4-digit (3-digit) enzyme classification (E.C.) numbers, native active site amino acids were generated at random for 472 of 550 cases. This increases to 548 of 550 cases when similar residues are allowed. The most frequently generated sequences correspond to ancient enzymatic functions, e.g., glycolysis, replication, and nucleotide biosynthesis. Surprisingly, even without selection, demi-chiral proteins possess the requisite marginal biochemical function and structure of modern proteins, but were thermodynamically less stable. If demi-chiral proteins were present, they could engage in early metabolism, which created the feedback loop for transcription and cell formation.

The phage display of Bacillus subtilis Lipase A significantly enhances catalytic activity due to altered nanoscale distribution in colloidal solution.

Screening libraries of mutant proteins by phage display is now relatively common. However, one unknown factor is how the bacteriophage scaffold itself influences the properties of the displayed protein. This communication evaluates the effect of solution parameters on the catalytic activity of phage displayed Bacillus subtilis Lipase A (BSLA), compared to the free enzyme in solution. While the pH- and temperature-activity profiles of BSLA were not intrinsically affected by phage display, the nanoscale distribution of BSLA within the micellar assay buffer was. This lead to a pronounced increase of activity of phage-BSLA relative to the free enzyme, owing to the accumulation of phage-BSLA at the substrate-rich micelles. Considering this result obtained for BSLA, caution is warranted and similar effects should be considered when selecting other enzymes/proteins by phage display, as the activity of the displayed protein may differ from that of the free protein.

Abstract DP-002: COMPARISON OF POTENTIAL OVARIAN CANCER BIOMARKERS BY MASS SPECTROMETRY-BASED PROTEOMIC ANALYSIS OF RESIDUAL PAP TEST FLUID, CERVICAL SWABS, AND TUMOR TISSUE FROM AN OVARIAN CANCER PATIENT

Abstract Early detection is the key to increased survival for women with ovarian cancer, yet a screening tool has yet to be developed that is adequately sensitive and specific enough for use in the general population. In contrast, screening for cervical cancer by Pap tests has been routinely performed for over 50 years. In the liquid-based Pap test, cells are collected from the cervix and placed into an alcohol-based fixative and then examined for abnormal cells. Since ovarian cancer cells have been observed in Pap tests, we reasoned that ovarian cancer peptide biomarkers may also be present. Our central hypothesis is that proteins shed by ovarian cancer cells can be detected during routine Pap tests by mass spectrometry (MS)-based proteomics. In particular, when collected at the time of cervical cancer Pap test screening, the alcohol-based Pap test fixative and cervical swabs are ideal for biomarker discovery since they are derived from a site near the ovarian cancer (i.e. proteins may be secreted or shed from the tumor and flow through the fallopian tube into the uterus and out the cervical opening). Recently, the fimbria of the fallopian tube have been suggested to be the true precursor to ovarian cancer, strengthening our hypothesis that ovarian cancer proteins will be found in the lower genital tract, perhaps even at early stages. To demonstrate the feasibility of using Pap tests as a biospecimen for proteomics, we previously examined the proteins present in residual Pap test fixative samples from women with normal cervical cytology by MS and described 152 proteins in the “Normal Pap Proteome.” The objective of this study was to identify and compare the proteins from three different sources from the same ovarian cancer patient: (i) the residual Pap test fixative, (ii) a Merocel swab of the cervix, and (iii) the primary ovarian cancer tumor tissue. Proteins were concentrated from the cell-free supernatant of the Pap test fixative or eluted from the swab, and then trypsin digested using the filter-aided sample preparation method. A total protein extract from the patient's tumor tissue was digested by standard in-solution trypsin digestion. The samples were run on 2D-liquid chromatography MS/MS, followed by bioinformatics integration. We identified over 5000 proteins total in the three samples. More than 2000 proteins were expressed in all three ovarian cancer samples, including several known ovarian cancer biomarkers such as CA125. By Scaffold analysis of the Gene Ontology nomenclature of the proteins, we classified the proteins by both cellular localization and biological processes. Additional matched samples from patients will be used to build a library of proteins and peptides that are specific to ovarian cancer for use in the development of targeted MS assays. We conclude that quantification of proteins from Pap test fixatives and cervical swabs will prove to be a rich source of biomarkers for ovarian cancer detection. Citation Format: Kristin L.M. Boylan, Anna C. Rogers, Melissa A. Geller, Peter A. Argenta, Timothy J. Griffin, and Amy P.N. Skubitz. COMPARISON OF POTENTIAL OVARIAN CANCER BIOMARKERS BY MASS SPECTROMETRY-BASED PROTEOMIC ANALYSIS OF RESIDUAL PAP TEST FLUID, CERVICAL SWABS, AND TUMOR TISSUE FROM AN OVARIAN CANCER PATIENT [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr DP-002.

Interactome Analysis of APOBEC3B in Multiple Myeloma

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) family proteins restrict retroviruses and retrotransposons by inducing hypermutation or degradation of the replication intermediates through their DNA cytidine deaminase activity. APOBECs can also act as endogenous sources of DNA damage that mutate many human cancers. Accumulation of APOBEC signature mutations is associated with disease progression and poor overall survival in multiple myeloma (Walker et al. Nat Commun, 2015). Among APOBEC3 enzymes, APOBEC3B (A3B) is the only family member that is predominantly located in the nucleus throughout the cell cycle. We previously reported that A3B knockdown decreased cytidine deaminase activity in myeloma cells, suggesting that among APOBECs, A3B plays a major role in cytidine deamination-related mutagenesis in myeloma cells (Yamazaki et al., Sci Rep, 2019). Recent studies showed that cofactors of A3B could affect the functions of A3B: heterogeneous nuclear ribonucleoproteins (hnRNPs) interact with surface hydrophobic residues of the N-terminal domain in order to bind to A3B (Xiao et al., Nuc. Acids Res, 2017; Zhang al., Cell Microbiol, 2008); BORF2, an Epstein-Barr viral protein, interacts with the A3B catalytic domain and inhibits A3B DNA cytidine deaminase activity (Cheng et al., Nat Microbiol, 2018). However, the biological mechanisms of how endogenous A3B induces mutations in genomic DNA are still unclear. In this study, we aim to ascertain the cofactors for nucleic acid binding and elucidate the regulatory mechanisms that prevent APOBEC-mediated genomic mutagenesis. Because of the high homology between APOBEC3 proteins, a specific antibody against A3B is not available, and it is difficult to analyze A3B-interaction at the endogenous expression level. To overcome this technical impediment, we used a lentiCRISPR system to insert a FLAG-tag sequence at the C-terminus of the A3B gene in A3B highly expressing myeloma cell lines (AMO1 and RPMI8226). We then conducted A3B-immunoprecipitation with the anti-FLAG M2 antibody, followed by mass spectrometry (MS) analysis to identify potential A3B interacting proteins. MS analysis identified 40 putative interacting proteins and these proteins were clustered largely into two interaction networks: ribonucleoprotein complex and ribosomal-associated proteins. We also performed Gene Ontology (GO) enrichment analysis and revealed that spliceosome, ribosome, and RNA transport were significantly enriched terms. We confirmed the binding between A3B and selected A3B interacting proteins: hnRNPs, interleukin enhancer-binding factor 2 and 3 (ILF2, ILF3) in myeloma cell lines by co-immunoprecipitation assays. Next, we tested the intracellular colocalization of overexpressed A3B and interacting proteins in Hela cells by immunofluorescence microscopy. We found that ILF2 presents strong colocalization with A3B in the nuclei of cells. We also employed density-gradient sedimentation analysis to test if these proteins form high molecular mass (HMM) complexes with A3B in the nucleus using HEK293T cells expressing FLAG-tagged A3B. We detected that ILF2 is one of the components of HMM A3B complexes. To check whether these putative interacting proteins affect A3B cytidine deaminase activity, we next performed an in vitro luminescence-based screening assay (AlphaScreen)using a FLAG-GST protein library which was produced by the wheat cell-free protein production system. We found that hnRNP A1 and ILF2 decreased A3B cytidine deaminase activity. This study provides for the first time a proteomic characterization of A3B interactome in a myeloma cell context. Our findings reveal putative A3B cofactors in myeloma cell lines which may regulate the catalytic activity of A3B. We discuss how these proteins bind A3B and affect its activity in myeloma cells. Disclosures Takaori-Kondo: Pfizer: Honoraria; Janssen: Honoraria; Novartis: Honoraria; Celgene: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ono: Research Funding; Takeda: Research Funding; Chugai: Research Funding; Kyowa Kirin: Research Funding.

Restriction-Free Construction of a Phage-Presented Very Short Macrocyclic Peptide Library.

Phage display is a commonly used technology for the screening of large clonal libraries of proteins and peptides. The construction of peptide libraries containing very short sequences, however, poses certain problems for conventional restriction-based cloning procedures, which are rooted in the necessity to purify restricted library oligos. Herein, we present an alternative cloning method especially suitable for such very short sequences of about only 21 base pairs resulting in a 60bp insert. The employed restriction-free hot fusion cloning strategy allows for facile library construction bypassing the need for purification of the small oligo. The library includes one well-defined disulfide bridge rendering the displayed macrocyclic peptide sequences as attractive scaffolds for novel active principles.

CDNA Display of Disulfide-Containing Peptide Library and In Vitro Evolution.

Directed in vitro evolution (IVE) is now a widely applied technology to obtain molecules that have designed new features of one's demands. We describe here experimental procedures for a cDNA display IVE to select peptide aptamers from a scaffold-based random peptide library. A three-finger (3-F) peptide library is exemplified, which has been shown its pluripotency to various target molecules. Peptide scaffolds including 3-F are refined through evolution, and they are mostly stabilized by disulfide bridges. To utilize such disulfide-containing protein library in IVE, we optimized the translation and folding conditions. Co-translational folding assisted by protein disulfide isomerase was found to have better efficiency than posttranslational refolding in the IVE. Linker is also a key element to make a tight genotype-phenotype linkage. Here, we introduced a whole procedure of IVE to use a newly designed puromycin linker, which was synthesized by a novel branching strategy. The improved linker enabled rapid and highly efficient ligation of mRNA and synthesis of protein fusions.

An improved platform for functional assessment of large protein libraries in mammalian cells.

Multiplex genetic assays can simultaneously test thousands of genetic variants for a property of interest. However, limitations of existing multiplex assay methods in cultured mammalian cells hinder the breadth, speed and scale of these experiments. Here, we describe a series of improvements that greatly enhance the capabilities of a Bxb1 recombinase-based landing pad system for conducting different types of multiplex genetic assays in various mammalian cell lines. We incorporate the landing pad into a lentiviral vector, easing the process of generating new landing pad cell lines. We also develop several new landing pad versions, including one where the Bxb1 recombinase is expressed from the landing pad itself, improving recombination efficiency more than 2-fold and permitting rapid prototyping of transgenic constructs. Other versions incorporate positive and negative selection markers that enable drug-based enrichment of recombinant cells, enabling the use of larger libraries and reducing costs. A version with dual convergent promoters allows enrichment of recombinant cells independent of transgene expression, permitting the assessment of libraries of transgenes that perturb cell growth and survival. Lastly, we demonstrate these improvements by assessing the effects of a combinatorial library of oncogenes and tumor suppressors on cell growth. Collectively, these advancements make multiplex genetic assays in diverse cultured cell lines easier, cheaper and more effective, facilitating future studies probing how proteins impact cell function, using transgenic variant libraries tested individually or in combination.

Membrane-associated RING-CH (MARCH) proteins down-regulate cell surface expression of the interleukin-6 receptor alpha chain (IL6Rα).

Interleukin 6 (IL6) is a cytokine that regulates a number of important immune and inflammatory pathways. We used the ability of IL6 to inhibit the clonal proliferation of the mouse M1 myeloid leukemia cell line in agar to positively screen a cDNA expression library for proteins that inhibited IL6 activity. We found three clones completely resistant to IL6 that contained the cDNA for the Membrane-Associated RING-CH E3 ubiquitin ligase MARCH2. MARCH2 is a member of a family of membrane-bound E3 ubiquitin ligases that target cell surface receptors for degradation. MARCH2 overexpressing M1 clones retained responsiveness to the related cytokines leukemia inhibitory factor and oncostatin M and we showed that its inhibitory effect was a result of selective down-regulation of the IL6 receptor alpha chain and not the shared receptor subunit, gp130 or other signalling molecules. This activity of MARCH2 was also shared with related proteins MARCH4, MARCH9 and an isoform of MARCH3. The transmembrane domains and C-terminal domains, as well as a functional RING domain, of MARCH proteins were all required for substrate recognition and down-regulation. Genetic deletion of individual MARCH proteins in mice had no or little effect on IL6Rα levels but combined deletions of MARCH2,3 and 4 displayed elevated steady-state levels of IL6Rα in selected haemopoietic cell subsets including CD8+ and CD4+ T cells. These studies extend the potential immunosuppressive roles of MARCH proteins to include down-regulation of IL6 inflammatory responses.

Proteomics analysis of serum protein patterns in duck during aflatoxin B1 exposure.

Background and Aim:Unlike the already well-documented human serum proteome, there are still limitations regarding analyzing and interpreting the various physiological changes and disease states of the serum proteomes found in duck. Serum proteome in duck under the condition of mycotoxin contamination in feed has not yet been examined. This study aimed to introduce the characterization of the circulating proteomes in duck serum during exposure to aflatoxin B1 (AFB1).Materials and Methods:Duck serum samples were collected from four experimental groups, gel-based mass spectrometry was then applied, and finally, 445 proteins were identified in pulled serum sample.Results:Among these 445 proteins, 377 were present in at least one group from all. There were 35 proteins which were expressed when the duck was exposed to AFB1. The protein library that allows the identification of a large number of different proteins in duck serum will be enhanced by the addition of these peptide spectral data. It is noteworthy that chromodomain-helicase-DNA-binding protein 7 (CHD7) [Gallus gallus] was up-regulated in the group with the highest AFB1 contamination.Conclusion:CHD7 protein might be somehow relative to aflatoxicosis in the duck that causes poor performance and economic loss. Moreover, other proteins present in duck serum were also added in the protein library.

Proteins mimicking epitope of HIV-1 virus neutralizing antibody induce virus-neutralizing sera in mice.

BackgroundThe development of an effective vaccine preventing HIV-1 infection is hindered by the enormous antigenic variability and unique biochemical and immunological properties of HIV-1 Env glycoprotein, the most promising target for HIV-1 neutralizing antibody. Functional studies of rare elite neutralizers led to the discovery of broadly neutralizing antibodies.MethodsWe employed a highly complex combinatorial protein library derived from a 5 kDa albumin-binding domain scaffold, fused with support protein of total 38 kDa, to screen for binders of broadly neutralizing antibody VRC01 paratope. The most specific binders were used for immunization of experimental mice to elicit Env-specific antibodies and to test their neutralization activity using a panel of HIV-1 clade C and B pseudoviruses.FindingsThree most specific binders designated as VRA017, VRA019, and VRA177 exhibited high specificity to VRC01 antibody. Immunized mice produced Env-binding antibodies which neutralize eight of twelve HIV-1 Tier 2 pseudoviruses. Molecular modelling revealed a shape complementarity between VRA proteins and a part of VRC01 gp120 interacting surface.InterpretationThis strategy based on the identification of protein replicas of broadly neutralizing antibody paratope represents a novel approach in HIV-1 vaccine development. This approach is not affected by low immunogenicity of neutralization-sensitive epitopes, variability, and unique biochemical properties of HIV-1 Env used as a crucial antigen in the majority of contemporary tested vaccines.FundCzech Health Research Council 15-32198A, Ministry of Health, Czech Republic.

Diselenide-selenoester ligation for chemical protein synthesis.

Chemoselective peptide ligation methods have provided synthetic access to numerous proteins, including those bearing native post-translational modifications and unnatural labels. This protocol outlines the chemical synthesis of proteins using a recently discovered reaction (diselenide-selenoester ligation (DSL)) in a rapid, additive-free manner. After ligation, the products can be chemoselectively deselenized to produce native peptide and protein products. We describe methods for the synthesis of suitably functionalized peptide diselenide and peptide selenoester fragments via Fmoc-solid-phase peptide synthesis (SPPS) protocols, fusion of these fragments by DSL, and the chemoselective deselenization of the ligation products to generate native synthetic proteins. We demonstrate the method's utility through the total chemical synthesis of the post-translationally modified collagenous domain of the hormone adiponectin via DSL-deselenization at selenocystine (the oxidized form of selenocysteine) and the rapid preparation of two tick-derived thrombin-inhibiting proteins by DSL-deselenization at β-selenoaspartate and γ-selenoglutamate. This method should find widespread use for the rapid synthesis of proteins, including cases in which other peptide ligation methods cannot be used (or cannot be used efficiently), e.g., at sterically hindered or deactivated acyl donors. The method's speed and efficiency may render it useful in the generation of synthetic protein libraries. Each protein discussed can be synthesized within 15 working days from resin loading and can be readily produced by practitioners with master's-level experience in organic chemistry. Each synthesis using these protocols was performed independently by two labs (one academic and one industrial), which attained comparable yields of the protein products.

Supervised Learning and Mass Spectrometry Predicts the in Vivo Fate of Nanomaterials.

The surface of nanoparticles changes immediately after intravenous injection because blood proteins adsorb on the surface. How this interface changes during circulation and its impact on nanoparticle distribution within the body is not understood. Here, we developed a workflow to show that the evolution of proteins on nanoparticle surfaces predicts the biological fate of nanoparticles in vivo. This workflow involves extracting nanoparticles at multiple time points from circulation, isolating the proteins off the surface and performing proteomic mass spectrometry. The mass spectrometry protein library served as inputs, while blood clearance and organ accumulation were used as outputs to train a supervised deep neural network that predicts nanoparticle biological fate. In a double-blinded study, we tested the network by predicting nanoparticle spleen and liver accumulation with upward of 94% accuracy. Our neural network discovered that the mechanism of liver and spleen uptake is due to patterns of a multitude of nanoparticle surface adsorbed proteins. There are too many combinations to change these proteins manually using chemical or biological inhibitors to alter clearance. Therefore, we developed a technique that uses the host to act as a bioreactor to prepare nanoparticles with predictable clearance patterns that reduce liver and spleen uptake by 50% and 70%, respectively. These techniques provide opportunities to both predict nanoparticle behavior and also to engineer surface chemistries that are specifically designed by the body.