Purification Of Fragments Research Articles

Proteomic analysis of the pyrenoid-traversing membranes of Chlamydomonas reinhardtii reveals novel components.

Pyrenoids are algal CO2-fixing organelles that mediate approximately one-third of global carbon fixation and hold the potential to enhance crop growth if engineered into land plants. Most pyrenoids are traversed by membranes that are thought to supply them with concentrated CO2. Despite the critical nature of these membranes for pyrenoid function, they are poorly understood, with few protein components known in any species.• Here we identify protein components of the pyrenoid-traversing membranes from the leading model alga Chlamydomonas reinhardtii by affinity purification and mass spectrometry of membrane fragments. Our proteome includes previously-known proteins as well as novel candidates.• We further characterize two of the novel pyrenoid-traversing membrane-resident proteins, Cre10.g452250, which we name Pyrenoid Membrane Enriched 1 (PME1), and LCI16. We confirm their localization, observe that they physically interact, and find that neither protein is required for normal membrane morphology.• Taken together, our study identifies the proteome of pyrenoid-traversing membranes and initiates the characterization of a novel pyrenoid-traversing membrane complex, building toward a mechanistic understanding of the pyrenoid.

Neutralizing anti-diphtheria toxin scFv produced by phage display.

Diphtheria can be prevented by vaccination, but some epidemics occur in several places, and diphtheria's threat is considerable. Administration of diphtheria antitoxin (DAT) produced from hyperimmunized animals is the most common treatment. Recombinant human antibody fragments such as single-chain variable fragments (scFv) produced by phage display library may introduce an interesting approach to overcome the limitations of the traditional antibody therapy. In the present study, B cells of immunized volunteers were used to construct a human single-chain fragment (HuscFv) library. The library was constructed with the maximum combination of heavy and light chains. As an antigen, Diphtheria toxoid (DTd) was used in four-round phage bio-panning to select phage clones that display DTd bound HuscFv from the library. After panning, individual scFv clones were selected. Clones that were able to detect DTd in an initial screening assay were transferred to Escherichia coli HB2151 to express the scFvs and purification was followed by Ni metal ion affinity chromatography. Toxin neutralization test was performed on Vero cells. The reactivity of the soluble scFv with diphtheria toxin were done and affinity calculation based on Beatty method was calculated. The size of the constructed scFv library was calculated to be 1.3×106 members. Following four rounds of selection, 40 antibody clones were isolated which showed positive reactivity with DTd in an ELISA assay. Five clones were able to neutralize DTd in Vero cell assay. These neutralizing clones were used for soluble expression and purification of scFv fragments. Some of these soluble scFv fragments show neutralizing activity ranging from 0.6 to 1.2µg against twofold cytotoxic dose of diphtheria toxin. The affinity constant of the selected scFv antibody was determined almost 107M-1. This study describes the prosperous construction and isolation of scFv from the immune library, which specifically neutralizes diphtheria toxin. The HuscFv produced in this study can be a potential candidate to substitute the animal antibody for treating diphtheria and detecting toxins.

ChEC-Seq: A Comprehensive Guide for Scalable and Cost-Efficient Genome-Wide Profiling in Saccharomyces cerevisiae.

Chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq) is a profiling method for protein-DNA interactions that can detect binding locations in vivo, does not require antibodies or fixation, and provides genome-wide coverage at near nucleotide resolution.The core of this method is an MNase fusion of the target protein, which allows it, when triggered by calcium exposure, to cut DNA at its binding sites and to generate small DNA fragments that can be readily separated from the rest of the genome and sequenced.Improvements since the original protocol have increased the ease, lowered the costs, and multiplied the throughput of this method to enable a scale and resolution of experiments not available with traditional methods such as ChIP-seq. This method describes each step from the initial creation and verification of the MNase-tagged yeast strains, over the ChEC MNase activation and small fragment purification procedure to the sequencing library preparation. It also briefly touches on the bioinformatic steps necessary to create meaningful genome-wide binding profiles.

Computational designing of the ligands of Protein L affinity chromatography based on molecular docking and molecular dynamics simulations

Protein L is a multidomain protein from Peptostreptococcus magnus with binding affinity to kappa light chain of human immunoglobulin (Ig) which is used for the purification of antibody fragments by affinity chromatography. The advances in protein engineering and computational biology approaches lead to the development of engineered affinity ligands with improved properties including binding affinity. In this study, molecular dynamics simulations (MDs) and Osprey software were used to design single B domains of the Protein L with higher affinity to antibody fragments. The modified B domains were then polymerized to ligand with six B domains by homology modeling methods. The results showed that single B domain mutants of MB1 (Thr865Trp) and MB2 (Thr847Met-Thr865Trp) had higher binding affinity to Fab compared to the wild single B domain. Also, MDs and molecular docking results showed that the polymerized Proteins L including the wild and mutated six B domains (6B0, 6B1, and 6B2) were stable during MDs and the two mutants of 6B1 and 6B2 showed higher binding affinity to Fab relative to the wild type. Communicated by Ramaswamy H. Sarma

Genome-wide identification and comprehensive analysis of WRKY transcription factor family in safflower during drought stress

The WRKY family is an important family of transcription factors in plant development and stress response. Currently, there are few reports on the WRKY gene family in safflower (Carthamus tinctorius L.). In this study, a total of 82 CtWRKY genes were identified from the safflower genome and could be classified into 3 major groups and 5 subgroups based on their structural and phylogenetic characteristics. The results of gene structure, conserved domain and motif analyses indicated that CtWRKYs within the same subfamily maintained a consistent exon/intron organization and composition. Chromosomal localization and gene duplication analysis results showed that CtWRKYs were randomly localized on 12 chromosomes and that fragment duplication and purification selection may have played an important role in the evolution of the WRKY gene family in safflower. Promoter cis-acting element analysis revealed that the CtWRKYs contain many abiotic stress response elements and hormone response elements. Transcriptome data and qRT-PCR analyses revealed that the expression of CtWRKYs showed tissue specificity and a strong response to drought stress. Notably, the expression level of the CtWRKY55 gene rapidly increased more than eightfold under drought treatment and rehydration, indicating that it may be a key gene in response to drought stress. These results provide useful insights for investigating the regulatory function of the CtWRKY gene in safflower growth and development, as well as identifying key genes for future molecular breeding programmes.

Determining the Hydrophobicity Index of Protected Amino Acids and Common Protecting Groups

Peptides are in great demand in the pharmaceutical arena and a majority of these peptides contain 20 or more amino acids. They are infrequently synthesised using the fragment condensation approach. A key limitation in adopting this approach more commonly is that protected peptide fragments with high purity are often required prior to the final condensation steps. It is hypothesized that understanding the hydrophobic nature of the protected amino acids will assist with designing optimal fragment purification processes when needed. Whilst a myriad of hydrophobicity indices are reported in the literature for unprotected amino acids, the literature lacks any data regarding the protected amino acids which form the key precursor for the fragment condensation task. In this current study, hydrophobicity indices for protected amino acids with common α-amino and sidechain protecting groups were experimentally determined. Different positions for each amino acid within the peptide chain were considered, namely at the C-terminal and N-terminal as well as internal positions. These data give deep insights on the hydrophobicity of each amino acid with respect to its position in the peptide chain. The data acquired in this research facilitated the prediction of the retention time of protected peptide fragments with an uncertainty of less than ±1.5%.

Functional fungal pellets self-immobilized by mycelium fragments of Irpex lacteus WRF-IL for efficient degradation of sulfamethazine as the sole carbon source

In order to achieve an efficient microbial material with dual functions of self-immobilization and sulfamethazine (SMZ) degradation, this study explored the pelletization technique utilizing mycelium fragments of Irpex lacteus WRF-IL and systematically examined the pellets formation conditions and degradation capability. The Box-Behnken design results demonstrated that pure mycelium fragments, broken by frosted glass beads, could be rapidly self-immobilized to form white rot mycelial pellets (WRMPs) within 24 h, serving as the pelleting core. These WRMPs could completely remove SMZ as the sole carbon source within 20 h. The addition of sucrose expedited this process, achieving complete removal within only 14 h. Kinetic analysis showed that WRMPs could potentially remove SMZ at higher concentrations (>25 mg/L). Biodegradation was the primary pathway of SMZ removal. Seven intermediates were identified by QTOF LC/MS, and three transformation pathways initiated by SO2 overflow, molecular rearrangement, and aniline moiety oxidation were deduced.

Calcium-dependent affinity ligands for the purification of antibody fragments at neutral pH

The emerging formats of antibody fragments for biotherapeutics suffer from inadequate purification methods, delaying the advances of innovative therapies. One of the top therapeutic candidates, the single-chain variable fragment (scFv), requires the development of individual purification protocols dependent on the type of scFv. The available approaches that are based on selective affinity chromatography but do not involve the use of a purification tag, such as Protein L and Protein A chromatography, require acidic elution buffers. These elution conditions can cause the formation of aggregates and thereby greatly compromise the yield, which can be a major problem for scFvs that are generally unstable molecules. Due to the costly and time-consuming production of biological drugs, like antibody fragments, we have engineered novel purification ligands that elute the scFvs in a calcium-dependent manner. The developed ligands are equipped with new, selective binding surfaces and were shown to efficiently elute all captured scFv at neutral pH with the use of a calcium chelator. Further, two of three ligands were proven not to bind to the CDRs of the scFv, indicating potential for use as generic affinity ligands to a range of different scFvs. Multimerization and optimization of the most promising ligand led to a 3-fold increase in binding capacity for the hexamer compared to the monomer, in addition to highly selective and efficient purification of a scFv with >95% purity in a single purification step. This calcium-dependent ligand could revolutionize the scFv industry, greatly facilitating the purification procedure and improving the quality of the final product.

Cloning, expression and purification of antigenic fragments of the Ureaplasma urealyticum UreD protein and its value in serology.

The detection of Ureaplasma urealyticum is usually done through culture. With the change of the smallest effective factor in culture, we face the lack of growth of these bacteria, which is one of the important reasons to find a suitable alternative for the diagnosis of this bacterium. UreD is a protected gene in this bacterium. The aim of this was to evaluate the ability of antigenic regions of UreD protein to bind to patients' serum antibodies. Antigenic regions of UreD protein were predicted using IEDB software with five different methods: Emini Surface Accessibility Prediction, Kolaskar and Tongaonkar Antigenicity, Chou and Fasman beta turn prediction, Karplus and Schulz flexibility scale, Ellipro-Epitope prediction based upon structural protrusion. Antigenic regions of UreD gene was clonned, expressed and purified. The antigenicity of this recombinant protein against the antibodies in the serum of people infected with U. urealyticum infections was checked in western blotting. The results showed that the antigenic regions of the UreD protein was producted and its antigenicity was demonstrated in western blotting. Moreover, all sera from patients infected with U. urealyticum reacted to the recombinant antigen. Specimens from people infected with U. urealyticum infection was positive in Western blotting suggesting that UreD protein has antigenic properties. Therefore, it can be used as a suitable candidate for the design of diagnostic kits and U. urealyticum vaccine.

Development of peptide affinity ligands for the purification of polyclonal and monoclonal Fabs from recombinant fluids

Engineered multi-specific monoclonal antibodies (msAbs) and antibody fragments offer valuable therapeutic options against metabolic disorders, aggressive cancers, and viral infections. The advancement in molecular design and recombinant expression of these next-generation drugs, however, is not equaled by the progress in downstream bioprocess technology. The purification of msAbs and fragments requires affinity adsorbents with orthogonal biorecognition of different portions of the antibody structure, namely its Fc (fragment crystallizable) and Fab (fragment antigen-binding) regions or the CH1-3 and CL chains. Current adsorbents rely on protein ligands that, while featuring high binding capacity and selectivity, need harsh elution conditions and suffer from high cost, limited biochemical stability, and potential release of immunogenic fragments. Responding to these challenges, we undertook the de novo discovery of peptide ligands that target different regions of human Fab and enable product release under mild conditions. The ligands were discovered by screening a focused library of 12-mer peptides against a feedstock comprising human Fab and Chinese hamster ovary host cell proteins (CHO HCPs). The identified ligands were evaluated via binding studies as well as molecular docking simulations, returning excellent values of binding capacity (Qmax ∼ 20 mg of Fab per mL of resin) and dissociation constant (KD = 2.16·10−6 M). Selected ligand FRWNFHRNTFFP and commercial Protein L ligands were further characterized by measuring the dynamic binding capacity (DBC10%) at different residence times (RT) and performing the purification of polyclonal and monoclonal Fabs from CHO-K1 cell culture fluids. The peptide ligand featured DBC10% ∼ 6-16 mg/mL (RT of 2 min) and afforded values of yield (93–96%) and purity (89–96%) comparable to those provided by Protein L resins.

Recombinant Protein L: Production, Purification and Characterization of a Universal Binding Ligand

Protein L (PpL) is a universal binding ligand that can be used for the detection and purification of antibodies and antibody fragments. Due to the unique interaction with immunoglobulin light chains, it differs from other affinity ligands, like protein A or G. However, due to its current higher market price, PpL is still scarce in applications. In this study, we investigated the recombinant production and purification of PpL and characterized the product in detail. We present a comprehensive roadmap for the production of the versatile protein PpL in E. coli.

Synthesis of the trisaccharide repeating unit of Stenotrophomonas maltophilia O6 antigen through step-wise and one-pot approaches

Synthetic conjugate vaccines are an important area of research for the prevention and occurrence of diseases caused by Gram-negative bacteria. For the development of such vaccines, access to the pure and homogeneous oligosaccharide fragments of the bacterial cell surface polysaccharides are necessary. Stenotrophomonas maltophilia is a typical opportunistic Gram-negative bacteria that causes severe pulmonary and other infections; often in hospitalized patients. With the emergence of multidrug resistant strains and increased virulence, new therapeutic strategies are needed to combat the threat. Herein, we report the syntheses of the trisaccharide repeating unit of S. maltophilia O6 antigen through stepwise and one-pot assemblies of the trisaccharide. The target trisaccharide was appended with a 2-aminoethyl linker that could provide the opportunity for conjugation to carrier proteins for the synthesis of vaccine candidates.

Analysis of chitosan molecular weight profile by electrophoresis in a porosity step gradient polyacrylamide gel

Chitosan is biocompatible and biodegradable natural biopolymer widely applied in various fields of biology, medicine, and pharmacy, however, its effects significantly depend on the degree of polymerization (DP) and the degree of deacetylation (DDA) of polymer chains. Evaluation of the chitosan chain diversity by DP requires the use of a highly expensive method of high-performance size exclusion chromatography. The aim of our study was to determine the molecular weight profile of chitosan specimens by the use of electrophoresis in a porosity step gradient polyacrylamide gel and to evaluate the efficacy of this method in monitoring the purification of chitosan fragments and its derivatives. Two types of step gradient porosity gels were used: 1) gels of layers with acrylamide concentration 2.5, 3.5, 5.0, 10.0, 15.0, 20.0 % w/v for native chitosan or its high molecular fragments; 2) gels of layers with acrylamide concentration 2.5, 5.0, 10.0, 15.0, 20.0, 25.0 % w/v for low molecular chitosan fragments. The main amount of molecules from the chitosan pool was localized in the type 1 gel in the region of 550-40 kDa and distributed among three bands, which in different samples differed significantly in percentage. Electrophoresis of chitosan fragments fractionated by gel permeation chromatography provided a clear separation of medium molecular weight fragments (50–400 kDa) in type 1 gel and of low molecular weight fragments (3–40 kDa) in type 2 gel. Thus the method of chitosan electrophoresis in a step-gradient porosity of polyacrylamide gel was developed which permits to characterize the molecular weight profile of chitosan specimens polymer chains and is effective in monitoring the isolation of chitosan fragments by gel penetration chromatography of molecular weights from 3 to 400 kDa.

Modelling the Photodegradation of Marine Microplastics by Means of Infrared Spectrometry and Chemometric Techniques

This work investigated the structural and chemical changes of plastics undergoing accelerated ageing upon irradiation that simulated the ultraviolet component of solar radiation for a five-year period. The plastics selected were polyethylene (PE) and polypropylene (PP) microplastic debris sampled from a sandy beach as well as pure pellets and fragments of objects made of the same polymers. We recorded Fourier Transform Infrared (FTIR) spectra at prescribed intervals during the irradiation procedure. The spectra were used to study the evolution of the absorption peaks usually associated with the environmental ageing of polyolefins, namely the peaks of hydroxyl and carbonyl stretching, the peaks relating to the presence of double bonds, and those associated with the crystallinity of PE and the tacticity of PP. The results showed that none of the usual degradation indexes followed a clear trend with increasing exposure and that the evolution of absorption peaks was not consistent among different fragments. We used the Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) method to process the whole information contained in the FTIR spectra in response to the chemical changes occurring during photochemical ageing. The results showed that the FTIR spectra contained sufficient information to cluster samples according to the irradiation received. Variable Importance of the Projection (VIP) analyses showed that the information for discriminating among different exposures was mainly contained in the absorption peaks corresponding to the hydroxyl and carbonyl stretching absorptions. The chemometric models had large determination coefficients, despite the large number of variables involved and could be applied to assess the environmental fate of plastics under environmental stressors.

A New Practice to Monitor the Fabrication Process of Fab-Targeting Ligands from Bevacizumab by LC-MS: Preparation and Analytical Characterization

The objective of this study was to qualitatively evaluate a Fab-targeting ligand preparation containing free thiol groups in the hinge region by using bevacizumab as a model. The evaluation focused on the purification of fragments through a nonaffinity-based process using a centrifugal ultrafiltration technique and mild reduction conditions for the intact production of F(ab’) fragments with specific inter-heavy-chain disulfide bonds cleavage. Under these conditions, F(ab’) fragments with a defined chemical composition were successfully obtained via proteolytic digestion followed by a controlled reduction reaction process maintaining the integrity of the binding sites. The ultrafiltration purification technique appears to be suitable for the removal of the digestive enzyme but inefficient for the removal of Fc fragments, thus requiring additional processing. A suitable analytical strategy was developed, allowing us to demonstrate the reformation of disulfide bridges between the two reduced cysteines within F(ab’) fragments.

Computational Engineering of Protein L to Achieve an Optimal Affinity Chromatography Resin for Purification of Antibody Fragments.

Protein L affinity chromatography is a useful method for the purification of antibody fragments containing kappa light chains. In affinity chromatography, increasing the binding affinity leads to increased product purity, recovery, and dynamic binding capacity (DBC). In this study, molecular docking and molecular dynamics simulation techniques were used to design the engineered Protein L with higher affinity to the kappa light chain. Each engineered ligand was produced as a recombinant protein and coupled to a solid matrix. The purity, recovery, and DBC of the engineered resins were evaluated and then compared to those of a commercially available resin. The results showed important parameters for engineering more efficient Protein L ligands for affinity chromatography.

Accumulation of endogenous and exogenous nucleic acids in "Touch DNA" components on hands.

Successful forensic DNA profiling from handled items is increasingly routine in casework. This "touch DNA" is thought to contain both cellular and acellular nucleic acid sources. However, there is little clarity on the origins or characteristics of this material. The cellular component consists of anucleate, terminally differentiated corneocytes (assumed to lack DNA), and the occasional nucleated cell. The acellular DNA source is fragmentary, presumably cell breakdown products. This study examines the relative contributions each component makes to the hand-secretions (endogenous) and hand-accumulations (exogenous) by recovering rinses from the inside and outside of worn gloves. Additionally, cellular and acellular DNA was measured at timepoints up to 2 h after hand washing, both with and without interim contact. Microscopic examination confirmed cell morphology and presence of nucleic acids. Following the novel application of a hair keratinocyte lysis method and plasma-DNA fragment purification to hand rinse samples, DNA profiles were generated from both fractions. Exogenous cell-free DNA is shown to be a significant source of touch DNA, which reaccumulates quickly, although its amplifiable nuclear alleles are limited. Endogenous DNA is mostly cellular in origin and provides more allelic information consistently over time.

Purification of antibody fragments via interaction with detergent micellar aggregates

The research described in this report seeks to present proof-of-concept for a novel and robust platform for purification of antibody fragments and to define and optimize the controlling parameters. Purification of antigen-binding F(ab′)2 fragments is achieved in the absence of chromatographic media or specific ligands, rather by using clusters of non-ionic detergent (e.g. Tween-60, Brij-O20) micelles chelated via Fe2+ ions and the hydrophobic chelator, bathophenanthroline (batho). These aggregates, quantitatively capture the F(ab′)2 fragment in the absence or presence of E. coli lysate and allow extraction of only the F(ab′)2 domain at pH 3.8 without concomitant aggregate dissolution or coextraction of bacterial impurities. Process yields range from 70 to 87% by densitometry. Recovered F(ab′)2 fragments are monomeric (by dynamic light scattering), preserve their secondary structure (by circular dichroism) and are as pure as those obtained via Protein A chromatography (from a mixture of F(ab′)2 and Fc fragments). The effect of process parameters on Ab binding and Ab extraction (e.g. temperature, pH, ionic strength, incubation time, composition of extraction buffer) are reported, using a monoclonal antibody (mAb) and polyclonal human IgG’s as test samples.

Strategies and Applications of Antigen-Binding Fragment (Fab) Production in Escherichia coli

AbstractWith the advancement of genetic engineering, monoclonal antibodies (mAbs) have made far-reaching progress in the treatment of various human diseases. However, due to the high cost of production, the increasing demands for antibody-based therapies have not been fully met. Currently, mAb-derived alternatives, such as antigen-binding fragments (Fab), single-chain variable fragments, bispecifics, nanobodies, and conjugated mAbs have emerged as promising new therapeutic modalities. They can be readily prepared in bacterial systems with well-established fermentation technology and ease of manipulation, leading to the reduction of overall cost. This review aims to shed light on the strategies to improve the expression, purification, and yield of Fab fragments in Escherichia coli expression systems, as well as current advances in the applications of Fab fragments.

Enrichment of Vascular Fragments from Mouse Embryonic Brains for Endothelial Cell Analysis

Endothelial cells in the brain interact with other cell types, forming the blood-brain barrier. This barrier controls the movement of solutes into and out of the brain, regulating pathophysiological processes and drug delivery to the brain. Common isolation methods used to study these cells during embryonic development involve enzymatic treatment and cell sorting using specific markers. This process modifies the cell state and produces minute amounts of sample. Here, we describe a protocol for the enrichment of vascular cells from embryonic brains based on dextran separation. In this method, the brain is lightly disrupted with a pestle and then resuspended in a dextran solution. Low-speed centrifugation permits the separation of the parenchymal and vascular fractions. Further centrifugation steps improve fractionation. This method is simple and fast and produces enough sample for biochemical assays. Graphic abstract: Purification of vascular fragments from an embryonic brain.