Acidic Isoelectric Point Research Articles

Modifications of DJ-1 in which pI shifts to acidic in red blood cells a potential biomarker for Parkinson’s disease at early stages

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases, the incidence of which increases with age. However, since there is no fundamental treatment or methods for early diagnosis, new methods of treatment and diagnosis are urgently needed. We focused on post-translational modifications of DJ-1, which is encoded by the familial PD-causative gene PARK7 in red blood cells (RBCs). DJ-1 has three cysteines (Cys46, Cys53, and Cys106), with Cys106 being preferentially oxidized. We previously reported that sulfinated/sulfonated Cys106 DJ-1 (oxDJ-1) is increased in the RBCs of PD patients. In this study, we analyzed RBC-derived DJ-1 from PD patients and control subjects by 2-dimensional electrophoresis. We found that the ratio of the spot of DJ-1 with a more acidic isoelectric point than oxDJ-1 was increased more significantly than that of oxDJ-1 in RBCs from patients at the early stage of unmedicated PD and decreased with the progression of PD stage and treatment. Furthermore, we revealed that this acidic spot of DJ-1 increased upon exposure to H2O2. However, when either Cys53 or Cys106 of DJ-1 was replaced with serine, there was no significant increase in the acidic spot caused by H2O2. In this study, we propose a new biomarker for early diagnosis of PD using both the ratios of oxDJ-1 to total DJ-1 and the acidic spot of DJ-1 to total DJ-1.

Different behavior of Ferguson plot between agarose and polyacrylamide gels

In this study, we conducted Ferguson plot analyses using both agarose and polyacrylamide gels in native electrophoresis and SDS-PAGE. The results revealed intriguing differences in the behavior of bovine serum albumin (BSA) and other model proteins. Specifically, BSA exhibited Ferguson plot slopes that were dependent on the oligomer size in agarose native gel electrophoresis, while such size-dependent behavior was not observed in native-PAGE or SDS-PAGE. These findings suggest that Ferguson plot analysis is a suitable approach when using agarose gel under the electrophoretic conditions employed in this study. Furthermore, our investigation extended to model proteins with acidic isoelectric points and larger molecular weights, namely Ferritin and caseinolytic peptidase B (ClpB). Notably, these proteins displayed distinct Ferguson plot slopes when subjected to agarose gel electrophoresis. Intriguingly, when polyacrylamide gel was employed, ClpB exhibited multiple bands, each with its unique Ferguson plot slope, deviating from the expected behavior based on molecular size. This divergence in Ferguson plot characteristics between agarose and polyacrylamide gels points to an interesting and complex interplay between protein properties and gel electrophoresis conditions.

A subfamily of the small heat shock proteins of the marine red alga Neopyropia yezoensis localizes in the chloroplast.

Small heat shock proteins (sHSPs) play a crucial role under abiotic stress and are present in all organisms, from eukaryotes to prokaryotes. However, studies on the sHSP gene family in red alga are limited. In this study, we aimed to identify and characterize NysHSP genes from the genome of N. yezoensis, a marine red alga adapted to the stressful intertidal zone. We identified seven NysHSP genes distributed on all three chromosomes. Expression analysis revealed that all NysHSP genes responded to H2O2 and heat stress in the gametophytic thalli, but these genes responded only to heat stress in the sporophytic conchocelis. NysHSP20.3, which has an acidic isoelectric point (pI) and short N-terminal region, was localized as granules in the cytosol. Fluorescence imaging of the NysHSP25.8-GFP and NysHSP28.4-GFP fusion proteins revealed that these proteins were located in the chloroplast. Based on their characteristics and cellular localization, the NysHSPs are divided into two subfamilies. Subfamily I includes four sHSP genes that strongly respond to heat stress and encode a protein localized in the cytosol. The NysHSP gene of subfamily II encodes a polypeptide with a long N-terminal region located in the chloroplast. This study provides insights into the evolution and function of the sHSP gene family of the marine red alga N. yezoensis and how it adapts to the stressful intertidal zone.

Oxidation affects dye binding of myofibrillar proteins via alteration in net charges mediated by a reduction in isoelectric point

In order to study the effect of oxidation on the dye-binding behavior of myofibrillar proteins, selected dyes with different charges (positively charged Sarfarin O (SO), neutral bromophenol blue (BPB), and negatively charged Orange G (OG)) were incubated with myofibrils oxidized by the Fenton system with H2O2 (10 mM). Upon oxidation, loss of free thiols, formation of carbonyls, particle size, and hydrophobicity of myofibrillar proteins (MPs) increased. The absolute value of Zeta-potential increased by 14.48 % after oxidation, the myofibrillar proteins shifted to a more acidic isoelectric point (pI) upon oxidation. Oxidation decreased net positive charges of myofibrillar protein and the binding ability of MPs towards OG in the environment with pH less than pI and the affinity of MPs towards SO in the environment with pH more than pI were thus increased. Here we propose a hypothesis that oxidation-induced change in net charges is the driving force affecting the amount of protein-bound dye. This paper aims to examine the effect of oxidation on the net charges of myofibrillar proteins and to provide insight into the mechanism of oxidation-induced changes in protein-bound dyes.

Heteroprotein complex coacervation of lactoferrin and osteopontin: Phase behaviour and thermodynamics of formation

This research describes the formation of reversible heteroprotein complex coacervates between the bioactive milk proteins lactoferrin (LF) and osteopontin (OPN) within specific combinations of pH (∼4–6), ionic strength (≤30 mM added NaCl), protein stoichiometry (LF:OPN mass mixing ratios of ∼ 2–8) and total protein concentration (≤∼8% w/v). Electrostatic attraction between these proteins is possible across a wide range of pH values due to the acidic isoelectric point of OPN (∼ pH 3.5) and the basic isoelectric point of LF (∼ pH 8.7). Thermodynamic analysis of the OPN-LF interaction indicated that the protein-protein binding had enthalpic and entropic contributions which resulted in liquid-liquid phase separation given the appropriate combination of intrinsic and extrinsic conditions. The complex coacervates were found to be a concentrated protein phase (∼27% protein, w/w) which was highly hydrated (>70% moisture, w/w) and occurred spontaneously within LF: OPN ratios naturally found in human milk. The structure, biological functionality, and applications of heteroprotein complexes and/or complex coacervates between LF and OPN will be explored in separate work.

Missense mutations in spike protein of SARS-CoV-2 delta variant contribute to the alteration in viral structure and interaction with hACE2 receptor.

IntroductionMany of the global pandemics threaten human existence over the decades among which coronavirus disease (COVID‐19) is the newest exposure circulating worldwide. The RNA encoded severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) virus is referred as the pivotal agent of this deadly disease that induces respiratory tract infection by interacting host ACE2 receptor with its spike glycoprotein. Rapidly evolving nature of this virus modified into new variants helps in perpetrating immune escape and protection against host defense mechanism. Consequently, a new isolate, delta variant originated from India is spreading perilously at a higher infection rate.MethodsIn this study, we focused to understand the conformational and functional significance of the missense mutations found in the spike glycoprotein of SARS‐CoV‐2 delta variant performing different computational analysis.ResultsFrom physiochemical analysis, we found that the acidic isoelectric point of the virus elevated to basic pH level due to the mutations. The targeted mutations were also found to change the interactive bonding pattern and conformational stability analyzed by the molecular dynamic's simulation. The molecular docking study also revealed that L452R and T478K mutations found in the RBD domain of delta variant spike protein contributed to alter interaction with the host ACE2 receptor.ConclusionsOverall, this study provided insightful evidence to understand the morphological and attributive impact of the mutations on SARS‐CoV‐2 delta variant.

Identification, characterization, and molecular phylogeny of scorpion enolase (Androctonus crassicauda and Hemiscorpius lepturus)

Scorpions are the oldest terrestrial fauna with an ancient evolutionary history. To study the origins of scorpions, we conducted a molecular phylogenetic study of the arthropod using slowly evolving enolase sequences. In this study, we used deep RNA-seq technology to identify the enolase sequences of Androctonus crassicauda and Hemiscorpius lepturus scorpions. The scorpion’s enolase sequences were in silico analyzed to predict the protein structures, physicochemical characterization, and exon-intron organization. The enolase of the scorpion was found to be a protein of 433 amino acids with a molecular mass of ∼47 kDa and an acidic isoelectric point. Here, we reported the existence of 8 exons and an inner GC/AG intron for scorpion enolase. Using homology modeling, the enolase of the scorpions was found to be very conservative during the evolution process. Furthermore, our phylogenetic analysis indicated the independent evolution of the Buthidae family from other scorpions and also the sister group relationship of Hemiscorpionidae with the Caraboctonidae families. The scorpions’ enolase sequences demonstrated a close fit to those of spiders, followed by mite counterparts. This strongly supports our phylogeny analysis, which indicates a sister-group relationship between scorpions and spiders and their common ancestors with mites. This study provided support for the pairing of scorpions with spiders, mites, etc. using enolase as a nuclear gene. Our molecular clock analyzes of the arthropod show that the most recent common ancestor of arachnids and insects were diversified in the Cryogenian period of Neoproterozoic era, earlier than dates reported from fossil records.

Cocaprins, β-Trefoil Fold Inhibitors of Cysteine and Aspartic Proteases from Coprinopsis cinerea.

We introduce a new family of fungal protease inhibitors with β-trefoil fold from the mushroom Coprinopsis cinerea, named cocaprins, which inhibit both cysteine and aspartic proteases. Two cocaprin-encoding genes are differentially expressed in fungal tissues. One is highly transcribed in vegetative mycelium and the other in the stipes of mature fruiting bodies. Cocaprins are small proteins (15 kDa) with acidic isoelectric points that form dimers. The three-dimensional structure of cocaprin 1 showed similarity to fungal β-trefoil lectins. Cocaprins inhibit plant C1 family cysteine proteases with Ki in the micromolar range, but do not inhibit the C13 family protease legumain, which distinguishes them from mycocypins. Cocaprins also inhibit the aspartic protease pepsin with Ki in the low micromolar range. Mutagenesis revealed that the β2-β3 loop is involved in the inhibition of cysteine proteases and that the inhibitory reactive sites for aspartic and cysteine proteases are located at different positions on the protein. Their biological function is thought to be the regulation of endogenous proteolytic activities or in defense against fungal antagonists. Cocaprins are the first characterized aspartic protease inhibitors with β-trefoil fold from fungi, and demonstrate the incredible plasticity of loop functionalization in fungal proteins with β-trefoil fold.

In silico structural, phylogenetic and drug target analysis of putrescine monooxygenase from Shewanella putrefaciens-95

BackgroundThe enormous and irresponsible use of antibiotics has led to the emergence of resistant strains of bacteria globally. A new approach to combat this crisis has been nutritional immunity limiting the availability of nutrients to pathogens. Targeting the siderophore biosynthetic pathway that helps in iron acquisition, an essential microelement in the bacterial system has been the topic of interest in recent days that backs the concept of nutritional immunity. Supporting this view, we have chosen to study a key enzyme in the biosynthetic pathway of putrebactin called putrescine monooxygenase (SpPMO) from Shewanella putrefaciens. In our previous study, we co-expressed putrescine monooxygenase recombinantly in Escherichia coli BL21 Star (DE3). The bioinformatic analysis and screening of inhibitors will broaden the scope of SpPMO as a drug target. ResultsIn the present study, we have analysed the physicochemical properties of the target enzyme and other N-hydroxylating monooxygenases (NMOs) using ExPASy server. The target enzyme SpPMO and most of the selected NMOs have a slightly acidic isoelectric point and are medially thermostable and generally insoluble. The multiple sequence alignment identified the GXGXX(N/A), DXXXFATGYXXXXP motives and conserved amino acids involved in FAD binding, NADP binding, secondary structure formation and substrate binding. The phylogenetic analysis indicated the distribution of the monooxygenases into different clades according to their substrate specificity. Further, a 3D model of SpPMO was predicted using I-TASSER online tool with DfoA from Erwinia amylovora as a template. The model was validated using the SAVES server and deposited to the Protein Model Database with the accession number PM0082222. The molecular docking analysis with different substrates revealed the presence of a putrescine binding pocket made of conserved amino acids and another binding pocket present on the surface of the protein wherein all other ligands interact with high binding affinity. The molecular docking of naturally occurring inhibitor molecules with SpPMO 3D model identified curcumin and niazirin with 1.83 and 2.81 μM inhibition constants as two promising inhibitors. Further studies on kinetic parameters of curcumin and niazirin inhibitors in vitro determined the Ki to be 2.6±0.0036 μM and 18.38±0.008 μM respectively. ConclusionThis analysis will help us understand the structural, phylogenetic and drug target aspects of putrescine monooxygenase from Shewanella putrefaciens-95 in detail. It sheds light on the precautionary measures that can be developed to inhibit the enzyme and thereby the secondary infections caused by them. Graphical abstract▪ Highlights•The physicochemical properties of putrescine monooxygenase from Shewanella putrefaciens-95 were compared with other known N-hydroxylating monooxygenases.•The phylogenetic analysis of SpPMO and other selected class B monooxygenases clearly classified NMOs according to their substrate affinity.•The molecular docking analysis of various substrates to SpPMO enzyme showed the presence of two binding pockets.•Curcumin and niazirin were identified as two potential natural inhibitors to SpPMO enzyme with 2.6±0.0036 μM and 18.38±0.008 μM inhibition constants respectively.

Expanding the range of sub/supercritical fluid chromatography: Advantageous use of methanesulfonic acid in water-rich modifiers for peptide analysis

The aim of this work was to expand the applicability range of UHPSFC to series of synthetic and commercialized peptides. Initially, a screening of different column chemistries available for UHPSFC analysis was performed, in combination with additives of either basic or acidic nature. The combination of an acidic additive (13 mM TFA) with a basic stationary phase (Torus DEA and 2-PIC) was found to be the best for a series of six synthetic peptides possessing either acidic, neutral or basic isoelectric points. Secondly, methanesulfonic acid (MSA) was evaluated as a potential replacement for TFA. Due to its stronger acidity, MSA gave better performance than TFA at the same concentration level. Furthermore, the use of reduced percentages of MSA, such as 8 mM, yielded similar results to those observed with 15 mM of MSA. The optimized UHPSFC method was, then, used to compare the performance of UHPSFC against RP-UHPLC for peptides with different pI and with increasing peptide chain length. UHPSFC was found to give a slightly better separation of the peptides according to their pI values, in few cases orthogonal to that observed in UHPLC. On the other hand, UHPSFC produced a much better separation of peptides with an increased amino acidic chain compared to UHPLC. Subsequently, UHPSFC-MS was systematically compared to UHPLC-MS using a set of linear and cyclic peptides commercially available. The optimized UHPSFC method was able to generate at least similar, and in some cases even better performance to UHPLC with the advantage of providing complementary information to that given by UHPLC analysis. Finally, the analytical UHPSFC method was transferred to a semipreparative scale using a proprietary cyclic peptide, demonstrating excellent purity and high yield in less than 15 min.

Acidic Solutions to Archaeal Chromatin

Cells must organize their genomes to provide compact structure and coordinate genome regulation. Like eukaryotes, most known archaea also encode histones, albeit with shorter sequences (∼68 amino acids) that span only the three-helix motif without additional domains. Most studies of histone structure-function relationships in archaea have focused on hyperthermophiles, such as Thermococcus kodakorensis and Methanothermus fervidus, where histones have been observed to wrap DNA into long super-helical ramps, referred to as archaeasomes. On the other hand, how histones function in other archaea is not well understood, especially in species that encode histones that have acidic isoelectric points. Normally, such acidic proteins would not be thought to bind nucleic acids. However, these histones maintain a basic ridge that may facilitate DNA binding despite their global acidic character. Here, we utilize biophysical assays and computational models to explore how acidic archaeal histones bind DNA and structure chromatin. Understanding how these archaeal species utilize acidic histones is essential for mapping how pressures from extreme environments has driven histone evolution and diversification.

Structure of human BCCIP and implications for binding and modification of partner proteins.

BCCIP was isolated based on its interactions with tumor suppressors BRCA2 and p21. Knockdown or knockout of BCCIP causes embryonic lethality in mice. BCCIP deficient cells exhibit impaired cell proliferation and chromosome instability. BCCIP also plays a key role in biogenesis of ribosome 60S subunits. BCCIP is conserved from yeast to humans, but it has no discernible sequence similarity to proteins of known structures. Here we report two crystal structures of an N-terminal truncated human BCCIPβ, consisting of residues 61-314. Structurally BCCIP is similar to GCN5-related acetyltransferases (GNATs) but contains different sequence motifs. Moreover, both acetyl-CoA and substrate-binding grooves are altered in BCCIP. A large 19-residue flap over the putative CoA binding site adopts either an open or closed conformation in BCCIP. The substrate binding groove is significantly reduced in size and is positively charged despite the acidic isoelectric point of BCCIP. BCCIP has potential binding sites for partner proteins and may have enzymatic activity.

Heterogeneity of Membrane Surface Proteins in Glanzmann’s Thrombasthenia

Studies in several laboratories have suggested that platelets from patients with Glanzmann’s thrombasthenia are deficient in two major membrane glycoproteins and that this membrane defect is uniform from patient to patient. We have used an improved electrophoretic technique to study further the surface composition of normal and thrombasthenie platelets. Platelets from three unrelated thrombasthenie patients were labeled by either lactoperoxidase-catalyzed iodination or the neuraminidase-galactose oxidase-[3H]NaBH4 technique and the labeled proteins were separated by two dimensional isoelectric focusing-SDS polyacrylamide gel electrophoresis. With both techniques, the major radiolabeled proteins were clearly separated from each other and were present as a horizontal collection of discrete spots that suggest charge heterogeneity. Most of the labeled proteins had an acidic isoelectric point. Compared to normal platelets, platelets from patients with Glanzmann’s disease contained no electrophoretically identifiable fibrinogen. In two patients with thrombasthenia, there was total absence of surface glycoproteins GPIIb and GPIII. while a third patient with thrombasthenia, who was clinically indistinguishable from the previous two patients, had decreased, but detectable, amounts of GPIIb and GPIII. These studies suggest that there are at least two phenotypic patterns of membrane abnormalities in Glanzmann’s thrombasthenia involving GPIIb and GPIII and may indicate genetic heterogeneity in this disease.

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

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

Identification of Indium Tin Oxide Nanoparticle-Binding Peptides via Phage Display and Biopanning Under Various Buffer Conditions.

By recent advances in phage-display approaches, many oligopeptides exhibiting binding affinities for metal oxides have been identified. Indium tin oxide is one of the most widely used conductive oxides, because it has a large band gap of 3.7-4.0 eV. In recent years, there have been reports about several ITO-based biosensors. Development of an ITO binding interface for the clustering of sensor proteins without complex bioconjugates is required. In this article, we aimed to identify peptides that bind to indium tin oxide nanoparticles via different binding mechanisms. Indium tin oxide nanoparticles binding peptide ware selected using phage display and biopanning against indium tin oxide, under five different buffer conditions and these peptides characterized about binding affinity and specificity. Three types of indium tin oxide nanoparticles-binding peptides were selected from 10 types of peptide candidates identified in phage display and biopanning. These included ITOBP8, which had an acidic isoelectric point, and was identified when a buffer containing guanidine was used, and ITOBP6 and ITOBP7, which contained a His-His-Lys sequence at their N-termini, and were identified when a highly concentrated phosphate elution buffer with a low ionic strength was used. Among these peptides, ITOBP6 exhibited the strongest indium tin oxide nanoparticlesbinding affinity (dissociation constant, 585 nmol/L; amount of protein bound at saturation, 17.5 nmol/m 2 - particles). These results indicate that peptides with specific binding properties can be obtained through careful selection of the buffer conditions in which the biopanning procedure is performed.

Structural characterization of the Pet c 1.0201 PR-10 protein isolated from roots of Petroselinum crispum (Mill.) Fuss

The native dimeric Petroselinum crispum (Mill.) Fuss protein Pet c 1.0201 and a monomeric xyloglucan endotransglycosylase enzyme (Garajova et al., 2008) isolated from the root cells co-purify and share similar molecular masses and acidic isoelectric points. In this work, we determined the complete primary structure of the parsley Pet c 1.0201 protein, based on tryptic and chymotryptic peptides followed by the manual micro-gradient chromatographic separation coupled with offline MALDI-TOF/TOF mass spectrometry. The bioinformatics approach enabled us to include the parsley protein into the PR-10 family, as it exhibited the highest protein sequence identity with the Apium graveolens Api g 1.0201 allergen and the major Daucus carota allergen Dau c 1.0201. Hence, we designated the Petroselinum crispum protein as Pet c 1.0201 and deposited it in the UniProt Knowledgebase under the accession C0HKF5. 3D protein homology modelling and molecular dynamics simulations of the Pet c 1.0201 dimer confirmed the typical structure of the Bet v 1 family allergens, and the potential of the Pet c 1.0201 protein to dimerize in water. However, the behavioural properties of Pet c 1.0201 and the celery allergen Api g 1.0101 differed in the presence of salts due to transiently and stably formed dimeric forms of Pet c 1.0201 and Api g 1.0101, respectively.

Studies on the Thiol Components of Isolated Nuclei

The thiol components of the nonhistone proteins prepared from isolated nuclei from rat liver, regenerating liver and hepatoma 223 cells have been investigated after reaction with radio labelled N-ethylmaleimide and 5-5’-dithiobis-(2-nitrobenzoic acid) (DTNB). The labelled adducts formed were examined by isoelectric focusing analysis in polyacrylamide gel and the distribution of the radiolabels within sliced portions of the gels determined. In the case of the 14C labelled NEM adduct the label was found to be spread amongst numerous protein components within the gel however, in the case of the 35S labelled DTNB adducts, only a small proportion of the label was found in the protein material which was retained in the acidic isoelectric point (pI) region of the gel. The bulk of the 35S labelled adduct (56% - 60%) was found to have migrated into the anode solution (10 mM phosphoric acid). This could be adsorbed onto a hydrophobic resin (XAD2) resin and eluted with methanol. Gel filtration chromatographic analysis of this adduct material on BioGel P2, (exclusion limit 1500 daltons) showed low molecular weight components to be present. Slightly different patterns were obtained for these nuclei, each containing several 35S components with molecular weights greater than the Ellman reagent itself. These 35S labelled thiol components did not contain any protein, peptide or amino acid components indicating strongly that a novel species of thiols could be present in these nuclei bound within the non-histone protein matrices.

Efficient profiling of detergent-assisted membrane proteome in cyanobacteria

Membrane proteins play key roles in cellular functions like transport of molecules, perception of environmental cues, and signal transduction into the intracellular compartment. However, the profiling of membrane proteins is still a daunting task because of the hydrophobicity, restricting our knowledge of membrane proteins. Thus, we attempted to develop a novel detergent-based approach to uncover membrane proteins using cyanobacteria. We investigated the effect of five different detergents on the profiling of the cyanobacterial membrane proteome. The application of either amidosulfobetaine-14 (ASB14) or N-lauroylsarcosine (NL) doubled the number of the identified integral membrane proteins compared with the control. Extraction with ASB14 increased the number of transmembrane helices over four times. The quantitative index (mol%) of membrane proteins also increased from 13 to 22% when ASB14, NL, and benzyldimethyl-n-hexadecylammonium chloride (BAC) were used. ASB14 treatment was particularly useful for identifying membrane proteins with higher molecular weights (Mr), and the addition of BAC to the cyanobacterial membrane could identify the membrane proteins with acidic isoelectric points (pI). To validate the efficiency of the detergent-based proteomics, the functional membrane protein complexes involved in energy metabolism were selected as an example and shown to be successful with the combined data of ASB14, NL, and zwittergent 3-10 (ZW3-10). Taken together, we suggest that the use of a specific detergent and the subsequent combination of proteome data is critical for detailed profiling of cyanobacterial functional membrane proteins.

Exploring the separation power of mixed-modal resins for purification of recombinant osteopontin from clarified Escherichia coli lysates.

Osteopontin (OPN) is a structural protein with potential value in therapeutic and diagnostic applications. Low titer, acidic isoelectric point, and the lack of well-defined secondary and tertiary structure were some of the challenges that complicated purification development of OPN from recombinant Escherichia coli lysates. Reported processes for OPN recovery from recombinant sources use nonorthogonal unit operations and often suffer from low yield. In this work, we expanded the search for an optimal OPN purification method by including mixed-modal resins with both ionic and hydrophobic properties (Capto adhere, HEA HyperCel, and PPA HyperCel). Plate-based high-throughput screening (HTS) platform revealed useful information about the interactions between the three different ligands and OPN as function of pH and ionic strength. The HTS data allowed the selection of OPN adsorption and elution conditions that were tested and optimized in a batch mode. In terms of purification factor and yield, HEA HyperCel performed significantly better than the other two mixed-modal resins. Pairing HEA HyperCel with a strong anion exchange step (Capto Q) resulted in a two-step purification process that achieved 45-fold purification of OPN with a final purity of 95% and 44% overall yield. The orthogonality provided by mixed-modal and ion exchange steps resulted in higher yield in fewer unit operations than reported processes. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2722, 2019.

Microbial Community Structure and Functional Potential Along a Hypersaline Gradient.

Salinity is one of the strongest environmental drivers of microbial evolution and community composition. Here we aimed to determine the impact of salt concentrations (2.5, 7.5, and 33.2%) on the microbial community structure of reclaimed saltern ponds near San Francisco, California, and to discover prospective enzymes with potential biotechnological applications. Community compositions were determined by 16S rRNA amplicon sequencing revealing both higher richness and evenness in the pond sediments compared to the water columns. Co-occurrence network analysis additionally uncovered the presence of microbial seed bank communities, potentially primed to respond to rapid changes in salinity. In addition, functional annotation of shotgun metagenomic DNA showed different capabilities if the microbial communities at different salinities for methanogenesis, amino acid metabolism, and carbohydrate-active enzymes. There was an overall shift with increasing salinity in the functional potential for starch degradation, and a decrease in degradation of cellulose and other oligosaccharides. Further, many carbohydrate-active enzymes identified have acidic isoelectric points that have potential biotechnological applications, including deconstruction of biofuel feedstocks under high ionic conditions. Metagenome-assembled genomes (MAGs) of individual halotolerant and halophilic microbes were binned revealing a variety of carbohydrate-degrading potential of individual pond inhabitants.