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At least 19 records

Simplification of the purification of heat stable recombinant low molecular weight proteins and peptides from GST-fusion products

The synthesis and purification of peptides of importance in the fields of research and medicine continue to be a challenging task. Chemical synthesis of oligopeptides, especially those greater than 25 amino acids, is cost prohibitive. On the other hand, several bottlenecks exist in the production of recombinant short peptides in heterologous expression hosts such as Escherichia coli (E. coli). In this paper, a rapid, cost-effective, and reliable method for the production and singlestep-purification of peptides and small proteins was developed. Five peptides and small proteins were overexpressed in E. coli as GST-fusion products in high yields. The recombinant peptides or proteins were successfully purified after enzymatic cleavage with selective heat-induced precipitation of the GST-affinity tag. Qualitative and quantitative analysis using SDS-PAGE and mass spectrometric methods suggest that the recombinant peptides/ proteins were purified to >95% homogeneity. Results of biophysical experiments, including multi-dimensional NMR spectroscopy, show that the purified proteins/ peptides retain their native conformation. Isothermal titration calorimetry studies indicate no significant change in the binding affinity of the heattreated purified product to their interacting partner(s) compared to the recombinant peptides purified by conventional chromatographic procedures without subjecting to heat treatment. In our opinion, the results reported render the purification of recombinant proteins/ peptides of biomedical relevance using our proposed method easy and reliable.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Detection of innate immune response modulating impurities (IIRMI) in therapeutic peptides and proteins: Impact of excipients

Unintended immunogenicity can affect the safety and efficacy of therapeutic proteins and peptides, so accurate assessments of immunogenicity risk can aid in the selection, development, and regulation of biologics. Product- and process- related impurities can act as adjuvants that activate the local or systemic innate immune response increasing the likelihood of product immunogenicity. Thus, assessing whether products have innate immune response modulating impurities (IIRMI) is a key component of immunogenicity risk assessments. Identifying trace levels of individual IIRMI can be difficult and testing individually for all potential impurities is not feasible. Therefore, to mitigate the risk, cell-based assays that use human blood cells or monocyte-macrophage reporter cell lines are being developed to detect minute quantities of impurities capable of eliciting innate immune activation. As these are cell-based assays, there is concern that excipients could blunt the cell responses, masking the presence of immunogenic IIRMI. Here, we explore the impact of frequently used excipients (non-ionic detergents, sugars, amino acids, bulking agents) on the sensitivity of reporter cell lines (THP-1- and RAW-Blue cells) and fresh human blood cells to detect purified TLR agonists as model IIRMI. We show that while excipients do not modulate the innate immune response elicited by TLR agonists in vivo, they can impact on the sensitivity of cell-based IIRMI assays. Reduced sensitivity to detect LPS, FSL-1, and other model IIRMI was also evident when testing 3 different recombinant drug products, product A (a representative mAb), B (a representative growth factor), C (a representative peptide), and their corresponding formulations. These results indicate that product formulations need to be considered when developing and validating cell-based assays for assessing clinically relevant levels of IIRMI in therapeutic proteins. Optimization of reporter cells, culture conditions and drug product concentration appear to be critical to minimize the impact of excipients and attain sensitive and reproducible assays.

60 APPLIED LIFE SCIENCES↗

Isoelectric focusing of proteins and peptides

Egg-white solution was chosen as the reference solution in order to assess the effects of operational parameters (voltage, flow rate, ampholine pH range and concentration, and protein concentration) of the RIEF apparatus on protein resolution. Topics of discussion include: (1) comparison of RIEF apparatus to conventional IEF techniques (column and PAG) with respect to resolution and throughput; (2) peptide and protein separation (AHF, Thymosin - Fraction 5, vasoactive peptide, L-asparaginase and ACP); and (3) detection of peptides - dansyl derivatives of amino acids and peptides, post-focusing fluorescent labeling of amino acids, peptides and proteins, and ampholine extraction from focused gels.

Egen, N.↗

Secondary structure determines electron transport in peptides

Proteins play a key role in biological electron transport, but the structure–function relationships governing the electronic properties of peptides are not fully understood. Despite recent progress, understanding the link between peptide conformational flexibility, hierarchical structures, and electron transport pathways has been challenging. Here, we use single-molecule experiments, molecular dynamics (MD) simulations, nonequilibrium Green’s function-density functional theory (NEGF-DFT), and unsupervised machine learning to understand the role of secondary structure on electron transport in peptides. Our results reveal a two-state molecular conductance behavior for peptides across several different amino acid sequences. MD simulations and Gaussian mixture modeling are used to show that this two-state molecular conductance behavior arises due to the conformational flexibility of peptide backbones, with a high-conductance state arising due to a more defined secondary structure (beta turn or 3 10 helices) and a low-conductance state occurring for extended peptide structures. These results highlight the importance of helical conformations on electron transport in peptides. Conformer selection for the peptide structures is rationalized using principal component analysis of intramolecular hydrogen bonding distances along peptide backbones. Molecular conformations from MD simulations are used to model charge transport in NEGF-DFT calculations, and the results are in reasonable qualitative agreement with experiments. Projected density of states calculations and molecular orbital visualizations are further used to understand the role of amino acid side chains on transport. Overall, our results show that secondary structure plays a key role in electron transport in peptides, which provides broad avenues for understanding the electronic properties of proteins.

Science & Technology - Other Topics↗

IsoMatchMS : Open-Source Software for Automated Annotation and Visualization of High Resolution MALDI-MS Spectra

Due to its speed, accuracy, and adaptability to various sample types, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has become a popular method to identify molecular isotope profiles from biological samples. Often MALDI-MS data do not include tandem MS fragmentation data, and thus the identification of compounds in samples requires external databases so that the accurate mass of detected signals can be matched to known molecular compounds. Most relevant MALDI-MS software tools developed to confirm compound identifications are focused on small molecules (e.g., metabolites, lipids) and cannot be easily adapted to protein data due to their more complex isotopic distributions. Here, we present an R package called IsoMatchMS for the automated annotation of MALDI-MS data for multiple datatypes: intact proteins, peptides, and glycans. This tool accepts already derived molecular formulas or, for proteomics applications, can derive molecular formulas from a list of input peptides or proteins including proteins with post-translational modifications. In conclusion, visualization of all matched isotopic profiles is provided in a highly accessible HTML format called a trelliscope display, which allows users to filter and sort by several parameters such as match scores and the number of peaks matched. IsoMatchMS simplifies the annotation and visualization of MALDI-MS data for downstream analyses.

47 OTHER INSTRUMENTATION↗

Programming Amphiphilic Peptoid Oligomers for Hierarchical Assembly and Inorganic Crystallization

Natural organisms make a wide variety of exquisitely complex, nano-, micro-, and macroscale structured materials in an energy-efficient and highly reproducible manner. During these processes, the information-carrying biomolecules (e.g., proteins, peptides, and carbohydrates) enable (1) hierarchical organization to assemble scaffold materials and execute high-level functions and (2) exquisite control over inorganic materials synthesis, generating biominerals whose properties are optimized for their functions. Inspired by nature, significant efforts have been devoted to developing functional materials that can rival those natural molecules by mimicking in vivo functions using engineered proteins, peptides, DNAs, sequence-defined synthetic molecules (e.g., peptoids), and other biomimetic polymers. Among them, peptoids, a new type of synthetic mimetics of peptides and proteins, have received particular attention because they combine the merits of both synthetic polymers (e.g., high chemical stability and efficient synthesis) and biomolecules (e.g., sequence programmability and biocompatibility). The lack of both chirality and hydrogen bonds in their backbone results in a highly designable peptoid-based system with reduced structural complexity and side chain-chemistry-dominated properties. Here in this Account, we present our recent efforts in this field by programming amphiphilic peptoid sequences for (1) the controlled self-assembly into different hierarchically structured nanomaterials with favorable properties and (2) manipulating inorganic (nano)crystal nucleation, growth, and assembly into superstructures. First, we designed a series of amphiphilic peptoids with controlled side chain chemistries that self-assembled into 1D highly stiff and dynamic nanotubes, 2D membrane-mimetic nanosheets, hexagonally patterned nanoribbons, and 3D nanoflowers. These crystalline nanostructures exhibited sequence-dependent properties and showed promise for different applications. The corresponding peptoid self-assembly pathways and mechanisms were also investigated by leveraging in situ atomic force microscopy studies and molecular dynamics simulations, which showed precise sequence dependency. Second, inspired by peptide- and protein-controlled formation of hierarchical inorganic nanostructures in nature, we developed peptoid-based biomimetic approaches for controlled synthesis of inorganic materials (e.g., noble metals and calcite), in which we took advantage of the substantial side chain chemistry of peptoids and investigated the relationship between the peptoid sequences and the morphology and growth kinetics of inorganic materials. For example, to overcome the challenges (e.g., complexity of protein- and peptide-folding, poor thermal and chemical stabilities) facing the area of protein- and peptide-controlled synthesis of inorganic materials, we recently reported the design of sequence-defined peptoids for controlled synthesis of highly branched plasmonic gold particles. Moreover, we developed a rule of thumb for designing peptoids that predictively enabled the morphological evolution from spherical to coral-shaped gold nanoparticles (NPs). With this Account, we hope to stimulate the research interest of chemists and materials scientists and promote the predictive synthesis of functional and robust materials through the design of sequence-defined synthetic molecules.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Delivered complementation in planta (DCIP) enables measurement of peptide-mediated protein delivery efficiency in plants

Abstract Using a fluorescence complementation assay, Delivered Complementation in Planta (DCIP), we demonstrate cell-penetrating peptide-mediated cytosolic delivery of peptides and recombinant proteins in Nicotiana benthamiana. We show that DCIP enables quantitative measurement of protein delivery efficiency and enables functional screening of cell-penetrating peptides for in-planta protein delivery. Finally, we demonstrate that DCIP detects cell-penetrating peptide-mediated delivery of recombinantly expressed proteins such as mCherry and Lifeact into intact leaves. We also demonstrate delivery of a recombinant plant transcription factor, WUSCHEL (AtWUS), into N. benthamiana. RT-qPCR analysis of AtWUS delivery in Arabidopsis seedlings also suggests delivered WUS can recapitulate transcriptional changes induced by overexpression of AtWUS. Taken together, our findings demonstrate that DCIP offers a new and powerful tool for interrogating cytosolic delivery of proteins in plants and highlights future avenues for engineering plant physiology.

59 BASIC BIOLOGICAL SCIENCES↗

Structure-based group A streptococcal vaccine design: Helical wheel homology predicts antibody cross-reactivity among streptococcal M protein–derived peptides

Group A streptococcus (Strep A) surface M protein, an α-helical coiled-coil dimer, is a vaccine target and a major determinant of streptococcal virulence. The sequence-variable N-terminal region of the M protein defines the M type and also contains epitopes that promote opsonophagocytic killing of streptococci. Recent reports have reported considerable cross-reactivity among different M types, suggesting the prospect of identifying cross-protective epitopes that would constitute a broadly protective multivalent vaccine against Strep A isolates. In this work, we have used a combination of immunological assays, structural biology, and cheminformatics to construct a recombinant M protein–based vaccine that included six Strep A M peptides that were predicted to elicit antisera that would cross-react with an additional 15 nonvaccine M types of Strep A. Rabbit antisera against this recombinant vaccine cross-reacted with 10 of the 15 nonvaccine M peptides. Two of the five nonvaccine M peptides that did not cross-react shared high sequence identity (≥50%) with the vaccine peptides, implying that high sequence identity alone was insufficient for cross-reactivity among the M peptides. Additional structural analyses revealed that the sequence identity at corresponding polar helical-wheel heptad sites between vaccine and nonvaccine peptides accurately distinguishes cross-reactive from non–cross-reactive peptides. On the basis of these observations, we developed a scoring algorithm based on the sequence identity at polar heptad sites. When applied to all epidemiologically important M types, this algorithm should enable the selection of a minimal number of M peptide–based vaccine candidates that elicit broadly protective immunity against Strep A.

60 APPLIED LIFE SCIENCES↗

Sel1-like proteins and peptides are the major Oxalobacter formigenes -derived factors stimulating oxalate transport by human intestinal epithelial cells

Kidney stones (KSs) are very common, excruciating, and associated with tremendous healthcare cost, chronic kidney disease (CKD), and kidney failure (KF). Most KSs are composed of calcium oxalate and small increases in urinary oxalate concentration significantly enhance the stone risk. Oxalate also potentially contributes to CKD progression, kidney disease-associated cardiovascular diseases, and poor renal allograft survival. This emphasizes the urgent need for plasma and urinary oxalate lowering therapies, which can be achieved by enhancing enteric oxalate secretion. We previously identified Oxalobacter formigenes (O. formigenes)-derived factors secreted in its culture-conditioned medium (CM), which stimulate oxalate transport by human intestinal Caco2-BBE (C2) cells and reduce urinary oxalate excretion in hyperoxaluric mice by enhancing colonic oxalate secretion. Given their remarkable therapeutic potential, we now identified Sel1-like proteins as the major O. formigenes-derived secreted factors using mass spectrometry and functional assays. Crystal structures for six proteins were determined to confirm structures and better understand functions. OxBSel1-14-derived small peptides P8 and P9 were identified as the major factors, with P8 + 9 closely recapitulating the CM’s effects, acting through the oxalate transporters SLC26A2 and SLC26A6 and PKA activation. Besides C2 cells, P8 + 9 also stimulate oxalate transport by human ileal and colonic organoids, confirming that they work in human tissues. Finally, P8 and P9 peptides are identified as the major O. formigenes-derived secreted factors and they have significant therapeutic potential for hyperoxalemia, hyperoxaluria, and related disorders, impacting the outcomes of patients suffering from KSs, enteric hyperoxaluria, primary hyperoxaluria, CKD, KF, and renal transplant recipients.

59 BASIC BIOLOGICAL SCIENCES↗

Binding mechanism and binding free energy of amino acids and citrate to hydroxyapatite surfaces as a function of crystallographic facet, pH, and electrolytes

Hydroxyapatite (HAP) is the major mineral phase in bone and teeth. The interaction of individual amino acids and citrate ions with different crystallographic HAP surfaces has remained uncertain for decades, creating a knowledge gap to rationally design interactions with peptides, proteins, and drugs. In this contribution, we quantify the binding mechanisms and binding free energies of the 20 end-capped natural amino acids and citrate ions on the basal (001) and prismatic (010)/(020) planes of hydroxyapatite at pH values of 7 and 5 for the first time at the molecular scale. We utilized over 1500 steered molecular dynamics simulations with highly accurate potentials that reproduce surface and hydration energies of (hkl) hydroxyapatite surfaces at different pH values. Charged residues demonstrate a much higher affinity to HAP than charge-neutral species due to the formation of superficial ion pairs and ease of penetration into layers of water molecules on the mineral surface. Binding free energies range from 0 to -60 kJ/mol and were determined with ~10% uncertainty. The highest affinity was found for citrate, followed by Asp(-) and Glu(-), and followed after a gap by Arg(+), Lys(+), as well as by His(+) at pH 5. The (hkl)-specific area density of calcium ions, the protonation state of phosphate ions, and subsurface directional order of the ions in HAP lead to surface-specific binding patterns. Amino acids without ionic side groups exhibit weak binding, between -3 and 0 kJ/mol, due to difficulties to penetrate the first layer of water molecules on the apatite surfaces. We explain recognition processes that remained elusive in experiments, in prior simulations, discuss agreement with available data, and reconcile conflicting interpretations. In conclusion, the findings can serve as useful input for the design of peptides, proteins, and drug molecules for the modification of bone and teeth-related materials, as well as control of apatite mineralization.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Hierarchical Materials from High Information Content Macromolecular Building Blocks: Construction, Dynamic Interventions, and Prediction

Hierarchical materials that exhibit order over multiple length scales are ubiquitous in nature. Because hierarchy gives rise to unique properties and functions, many have sought inspiration from nature when designing and fabricating hierarchical matter. More and more, however, nature’s own high-information content building blocks, proteins, peptides, and peptidomimetics, are being coopted to build hierarchy because the information that determines structure, function, and interfacial interactions can be readily encoded in these versatile macromolecules. Here, we take stock of recent progress in the rational design and characterization of hierarchical materials produced from high-information content blocks with a focus on stimuli-responsive and “smart” architectures. We also review advances in the use of computational simulations and data-driven predictions to shed light on how the side chain chemistry and conformational flexibility of macromolecular blocks drive the emergence of order and the acquisition of hierarchy and also on how ionic, solvent, and surface effects influence the outcomes of assembly. Furthermore, continued progress in the above areas will ultimately usher in an era where an understanding of designed interactions, surface effects, and solution conditions can be harnessed to achieve predictive materials synthesis across scale and drive emergent phenomena in the self-assembly and reconfiguration of high-information content building blocks.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Rate constants measured for hydrated electron reactions with peptides and proteins

Effects of ionizing radiation on the amino acids of proteins and the reactivity of the protonated amino group depends upon the pK subscript a of the group. Estimates of the rate constants for reactions involving the amino acid side chains are presented. These rate constants gave an approximate rate constant for three different protein molecules.

Braams, R.↗

Formation of stable submicron peptide or protein particles by thin film freezing

The present invention includes compositions and methods for preparing micron-sized or submicron-sized particles by dissolving a water soluble effective ingredient in one or more solvents; spraying or dripping droplets solvent such that the effective ingredient is exposed to a vapor-liquid interface of less than 50, 100, 150, 200, 250, 200, 400 or 500 cm−1 area/volume to, e.g., increase protein stability; and contacting the droplet with a freezing surface that has a temperature differential of at least 30° C. between the droplet and the surface, wherein the surface freezes the droplet into a thin film with a thickness of less than 500 micrometers and a surface area to volume between 25 to 500 cm−1.

Johnston, Keith P.↗