Purification Protocol Research Articles

Expression and purification of active human 17β-Hydroxysteroid dehydrogenase type 1 from Escherichia coli.

Breast cancer cell growth is often dependent on the presence of steroidal hormones. The 17β-hydroxysteroid dehydrogenase type 1 isoform (17βHSD1) catalyzes NADPH-dependent conversion of estrone to estradiol, a more potent estrogen, and represents potential drug target for breast cancer treatment. To provide active enzyme for inhibitor screening, 17βHSD1 is usually expressed in insect or mammalian cells, or isolated from human placenta. In the present study we describe a simple protocol for expression and purification of active human 17βHSD1 from BL21(DE3) Escherichia coli cells. Soluble human 17βHSD1 was expressed using a pET28a(+)-based plasmid, which encodes a hexahistidine tag fused to the N-terminus of the protein, and purified by nickel affinity chromatography. The enzyme activity of purified 17βHSD1 was verified by three methods: thin-layer chromatography, an alkali assay and a spectroscopic assay. These non-radioactive enzyme assays require only standard laboratory equipment, and can be used for screening compounds that modulate 17βHSD1 activity.

A protocol for anti-CD4 IgG antibody purification using plasma samples from people with HIV and antibody-mediated cytotoxicity

Background. Up to 20% of people with HIV (PWH) fail to recover their CD4+ T cell counts to levels similar to healthy controls after suppressive antiretroviral therapy (ART). Immune non-responders (INRs) are PWH on suppressive ART with CD4+ T cell counts lower than 350 cells/mL, whereas their CD8+ T cell counts are higher than healthy controls. We are the first group to report that increased anti-CD4 autoantibody IgGs in INRs are responsible for blunted CD4+ T cell reconstitution in PWH with ART and viral suppression through anti-CD4 IgG-induced antibody-mediated cytotoxicity (ADCC) against CD4+ T cells in vitro. Notably, anti-CD4 IgG-mediated poor CD4+ T cell recovery from suppressive ART is the only mechanism targeting CD4+ T cells, specifically. Results. We provide a detailed one-by-one step protocol from antigen-specific antibody isolation using plasma samples, to ADCC assay. Conclusions. To promote reproducible research, a detailed protocol for isolating anti-CD4 IgG autoantibodies from plasma samples of PWH and evaluating ADCC effects is reported here.•Antigen-specific antibody isolation using human plasma samples•Antibody-mediated cytotoxicity (ADCC)

Purification of Photorhabdus Virulence Cassette (PVC) Protein Complexes from Escherichia coli for Artificial Translocation of Heterologous Cargo Proteins.

Contractile injection systems (CISs), one of the most important bacterial secretion systems that transport substrates across the membrane, are a collection of diverse but evolutionarily related macromolecular devices. Numerous effector proteins can be loaded and injected by this secretion complex to their specific destinations. One group of CISs called extracellular CIS (eCIS) has been proposed as secretory molecules that can be released from the bacterial cytoplasm and attack neighboring target cells from the extracellular environment. This makes them a potential delivery vector for the transportation of various cargos without the inclusion of bacterial cells, which might elicit certain immunological responses from hosts. We have demonstrated that the Photorhabdus virulence cassette (PVC), which is a typical eCIS, could be applied as an ideal vector for the translocation of proteinaceous cargos with different physical or chemical properties. Here, we describe the in-depth purification protocol of this mega complex from Escherichia coli. The protocol provided is a simpler, faster, and more productive way of generating the eCIS complexes than available methodologies reported previously, which can facilitate the subsequent applications of these nanodevices and other eCIS in different backgrounds.

Investigation of long non-coding RNAs in extracellular vesicles from low-volume blood serum specimens of colorectal cancer patients.

Colorectal cancer (CRC) is the second most prevalent cancer type worldwide, which highlights the urgent need for non-invasive biomarkers for its early detection and improved prognosis. We aimed to investigate the patterns of long non-coding RNAs (lncRNAs) in small extracellular vesicles (sEVs) collected from low-volume blood serum specimens of CRC patients, focusing on their potential as diagnostic biomarkers. Our research comprised two phases: an initial exploratory phase involving RNA sequencing of sEVs from 76 CRC patients and 29 healthy controls, and a subsequent validation phase with a larger cohort of 159 CRC patients and 138 healthy controls. Techniques such as dynamic light scattering, transmission electron microscopy, and Western blotting were utilized for sEV characterization. Optimized protocol for sEV purification, RNA isolation and preamplification was applied to successfully sequence the RNA content of sEVs and validate the results by RT-qPCR. We successfully isolated sEVs from blood serum and prepared sequencing libraries from a low amount of RNA. High-throughput sequencing identified differential levels of 460 transcripts between CRC patients and healthy controls, including mRNAs, lncRNAs, and pseudogenes, with approximately 20% being lncRNAs, highlighting several tumor-specific lncRNAs that have not been associated with CRC development and progression. The validation phase confirmed the upregulation of three lncRNAs (NALT1, AL096828, and LINC01637) in blood serum of CRC patients. This study not only identified lncRNA profiles in a population of sEVs from low-volume blood serum specimens of CRC patients but also highlights the value of innovative techniques in biomolecular research, particularly for the detection and analysis of low-abundance biomolecules in clinical samples. The identification of specific lncRNAs associated with CRC provides a foundation for future research into their functional roles in cancer development and potential clinical applications.

Isolation of Cardanol Fractions from Cashew Nutshell Liquid (CNSL): A Sustainable Approach

Exploring sustainable approaches to replace petroleum-based chemicals is an ongoing challenge in reducing the carbon footprint. Due to the complexity and percentage variation in nature-generated molecules, which further varies based on geographical origin and the purification protocol adopted, a better isolation strategy for individual components is required. Agrowaste from the cashew industry generates phenolic lipid (cardanol)-rich cashew nutshell liquid (CNSL) and has recently shown extensive commercial utility. Cardanol naturally exists as a mixture of three structurally different components with C15-alkylene chains: monoene, diene, and triene. The separation of these three fractions has been a bottleneck and is crucial for certain structural designs and reproducibility. Herein, we describe the gram-scale purification of cardanol into each component using flash column chromatography within the sustainability framework. The solvent used for elution is recovered and reused after each stage (up to 82%), making it a cost-effective and sustainable purification strategy. This simple purification technique replaces the alternative high-temperature vacuum distillation, which requires substantial energy consumption and poses vacuum fluctuation and maintenance challenges. Three components (monoene 42%, diene 22%, and triene 36%) were isolated with good purity and were fully characterized by 1H and 13C NMR, GC-MS, HPLC, and FTIR spectroscopy. The present work demonstrates that greener and simpler strategies pave the way for the isolation of constituents from nature-sourced biochemicals and unleash the potential of CNSL-derived fractions for high-end applications.

A rapid one-step affinity purification of C-phycocyanin from Spirulina platensis

The high-purity phycocyanin has a high commercial value. Most current purification methods of C-phycocyanin involve multiple steps, which are complicated and time-consuming. To solve the problem, this research was studied, and an efficient affinity chromatography purification for C-phycocyanin from Spirulina platensis was developed. Through molecular docking simulation, virtual screening of ligands was performed, and ursolic acid was identified as the specific affinity ligand, which coupled to Affi-Gel 102 gel via 1-ethyl (3-dimethylaminopropyl)-3-carbodiimide, hydrochloride as coupling agent. With this customized and synthesized resin, a high-efficiency one-step purification procedure for C-phycocyanin was developed and optimized, the purity was determined to be 4.53, and the yield was 69 %. This one-step purification protocol provides a new approach for purifying other phycobilin proteins.

A protocol for cloning, expression, and purification of Lysine exporter (LysE) gene of Mycobacterium tuberculosis

Background Tuberculosis (TB) is among the deadliest diseases and a significant cause of illnessacross the globe. Several studies on mycobacterial proteins, such as proteases and transporters that are essential for survival and pathogenesis have aimed to develop an efficient anti-tubercular agent. In mycobacterium, lysine exporter (LysE) is an amino acid transporter and a probable target for an anti-tubercular agent as it is responsible for bacterial growth inhibition and is also absent in the widely used Bacillus Calmette-Guérin (BCG) vaccine. Methods Some studies have purified LysE using different protocols. This study describes a protocol for purifying different constructs of LysE, focusing on its hydrophobic region using immobilized metal affinity chromatography (IMAC) after expressing LysE gene in a bacterial expression system. pET vector (pET28a) is used as an expression vector. Amplified LysE gene is ligated with the pET28a vector, and the resultant plasmid is then transformed into E. coli cells. The vector has a histidine tag that makes the purification process convenient. After IMAC, the samples will be subjected to size-exclusion chromatography for further purification. Results Cloning and amplification findings will be analyzed using 1% agarose gel, and protein expression and purification outcomes will be examined using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Domain-specific constructs of LysE can be further analyzed as an anti-tubercular agent. Conclusions Despite being a potential anti-tubercular target, research is quite limited on this protein. Therefore, we aim to purify LysE protein for further analysis. Similar protocols have already been implemented to purify several other bacterial proteins with >95% purity.

Scaling up production of recombinant human basic fibroblast growth factor in an Escherichia coli BL21(DE3) plysS strain and evaluation of its pro-wound healing efficacy.

Introduction: Human basic fibroblast growth factor (hbFGF) is a highly valuable multifunctional protein that plays a crucial role in various biological processes. In this study, we aim to accomplish the scaling-up production of mature hbFGF (146aa) by implementing a high cell-density fermentation and purification process on a 500-L scale, thereby satisfying the escalating demands for both experimental research and clinical applications. Methods: The hbFGF DNA fragment was cloned into a mpET-3c vector containing a kanamycin resistance gene and then inserted into Escherichia coli BL21 (DE3) plysS strain. To optimize the yield of hbFGF protein, various fermentation parameters were systematically optimized using BOX-Behnken design and further validated in large-scale fermentation (500-L). Additionally, a three-step purification protocol involving CM-Sepharose, heparin affinity, and SP-Sepharose column chromatography was developed to separate and purify the hbFGF protein. Isoelectric focusing electrophoresis, MALDI-TOF/MS analysis, amino acid sequencing, CD spectroscopy, and Western blotting were performed to authenticate its identity. The biological efficacy of purified hbFGF was evaluated using an MTT assay as well as in a diabetic deep second-degree scald model. Results: The engineered strain was successfully constructed, exhibiting high expression of hbFGF and excellent stability. Under the optimized fermentation conditions, an impressive bacterial yield of 46.8 ± 0.3g/L culture with an expression level of hbFGF reaching 28.2% ± 0.2% was achieved in 500-L scale fermentation. Subsequently, during pilot-scale purification, the final yield of purified hbFGF protein was 114.6 ± 5.9mg/L culture with RP-HPLC, SEC-HPLC, and SDS-PAGE purity exceeding 98%. The properties of purified hbFGF including its molecular weight, isoelectric point (pI), amino sequence, and secondary structure were found to be consistent with theoretical values. Furthermore, the purified hbFGF exhibited potent mitogenic activity with a specific value of 1.05 ± 0.94 × 106AU/mg and significantly enhanced wound healing in a deep second-degree scald wound diabetic rat model. Conclusion: This study successfully established a stable and efficient large-scale production process of hbFGF, providing a solid foundation for future industrial production.

An Efficient Expression and Purification Protocol for SpCas9 Nuclease and Evaluation of Different Delivery Methods of Ribonucleoprotein.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system is a revolutionary tool for precise genome editing across various cell types. Ribonucleoproteins (RNPs), encompassing the Cas9 protein and guide RNA (gRNA), have emerged as a promising technique due to their increased specificity and reduced off-target effects. This method eliminates the need for plasmid DNA introduction, thereby preventing potential integration of foreign DNA into the target cell genome. Given the requirement for large quantities of highly purified protein in various Cas9 studies, we present an efficient and simple method for the preparation of recombinant Streptococcus pyogenes Cas9 (SpCas9) protein. This method leverages the Small Ubiquitin Like Modifier(SUMO) tag system, which includes metal-affinity chromatography followed by anion-exchange chromatography purification. Furthermore, we compare two methods of CRISPR-Cas9 system delivery into cells: transfection with plasmid DNA encoding the CRISPR-Cas9 system and RNP transfection with the Cas9-gRNA complex. We estimate the efficiency of genomic editing and protein lifespan post-transfection. Intriguingly, we found that RNP treatment of cells, even in the absence of a transfection system, is a relatively efficient method for RNP delivery into cell culture. This discovery is particularly promising as it can significantly reduce cytotoxicity, which is crucial for certain cell cultures such as induced pluripotent stem cells (iPSCs).

A Tandem-Affinity Purification Method for Identification of Primary Intracellular Drug-Binding Proteins.

In the field of drug discovery, understanding how small molecule drugs interact with cellular components is crucial. Our study introduces a novel methodology to uncover primary drug targets using Tandem Affinity Purification for identification of Drug-Binding Proteins (TAP-DBP). Central to our approach is the generation of a FLAG-hemagglutinin (HA)-tagged chimeric protein featuring the FKBP12(F36V) adaptor protein and the TurboID enzyme. Conjugation of drug molecules with the FKBP12(F36V) ligand allows for the coordinated recruitment of drug-binding partners effectively enabling in-cell TurboID-mediated biotinylation. By employing a tandem affinity purification protocol based on FLAG-immunoprecipitation and streptavidin pulldown, alongside mass spectrometry analysis, TAP-DBP allows for the precise identification of drug-primary binding partners. Overall, this study introduces a systematic, unbiased method for identification of drug-protein interactions, contributing a clear understanding of target engagement and drug selectivity to advance the mode of action of a drug in cells.

Expression and purification of the receptor-binding domain of SARS-CoV-2 spike protein in mammalian cells for immunological assays.

The receptor-binding domain (RBD) of the spike glycoprotein of SARS-CoV-2 virus mediates the interaction with the host cell and is required for virus internalization. It is, therefore, the primary target of neutralizing antibodies. The receptor-binding domain soon became the major target for COVID-19 research and the development of diagnostic tools and new-generation vaccines. Here, we provide a detailed protocol for high-yield expression and one-step affinity purification of recombinant RBD from transiently transfected Expi293F cells. Expi293F mammalian cells can be grown to extremely high densities in a specially formulated serum-free medium in suspension cultures, which makes them an excellent tool for secreted protein production. The highly purified RBD is glycosylated, structurally intact, and forms homomeric complexes. With this quick and easy method, we are able to produce large quantities of RBD (80 mg·L-1 culture) that we have successfully used in immunological assays to examine antibody titers and seroconversion after mRNA-based vaccination of mice.

Entanglement Purification with Quantum LDPC Codes and Iterative Decoding

Recent constructions of quantum low-density parity-check (QLDPC) codes provide optimal scaling of the number of logical qubits and the minimum distance in terms of the code length, thereby opening the door to fault-tolerant quantum systems with minimal resource overhead. However, the hardware path from nearest-neighbor-connection-based topological codes to long-range-interaction-demanding QLDPC codes is likely a challenging one. Given the practical difficulty in building a monolithic architecture for quantum systems, such as computers, based on optimal QLDPC codes, it is worth considering a distributed implementation of such codes over a network of interconnected medium-sized quantum processors. In such a setting, all syndrome measurements and logical operations must be performed through the use of high-fidelity shared entangled states between the processing nodes. Since probabilistic many-to-1 distillation schemes for purifying entanglement are inefficient, we investigate quantum error correction based entanglement purification in this work. Specifically, we employ QLDPC codes to distill GHZ states, as the resulting high-fidelity logical GHZ states can interact directly with the code used to perform distributed quantum computing (DQC), e.g. for fault-tolerant Steane syndrome extraction. This protocol is applicable beyond the application of DQC since entanglement distribution and purification is a quintessential task of any quantum network. We use the min-sum algorithm (MSA) based iterative decoder with a sequential schedule for distilling 3-qubit GHZ states using a rate 0.118 family of lifted product QLDPC codes and obtain an input fidelity threshold of ≈0.7974 under i.i.d. single-qubit depolarizing noise. This represents the best threshold for a yield of 0.118 for any GHZ purification protocol. Our results apply to larger size GHZ states as well, where we extend our technical result about a measurement property of 3-qubit GHZ states to construct a scalable GHZ purification protocol.

Enzymatic characterization and application of soybean hull peroxidase as an efficient and renewable biocatalyst for degradation of zearalenone

Zearalenone (ZEN) is a notorious mycotoxin commonly found in Fusarium-contaminated crops, which causes great loss in livestock farming and serious health problems to humans. In the present work, we found that crude peroxidase extraction from soybean hulls could use H2O2 as a co-substate to oxidize ZEN. Molecular docking and dynamic simulation also supported that ZEN could bind to the active site of soybean hull peroxidase (SHP). Subsequently, SHP extracted from soybean hulls was purified using a combined purification protocol involving ammonium sulfate precipitation, ion exchange chromatography and size exclusion chromatography. The purified SHP showed wide pH resistance and high thermal stability. This peroxidase could degrade 95 % of ZEN in buffer with stepwise addition of 100 μM H2O2 in 1 h. The two main ZEN degradation products were identified as 13-OH-ZEN and 13-OH-ZEN-quinone. Moreover, SHP-catalyzed ZEN degradation products displayed much less cytotoxicity to human liver cells than ZEN. The application of SHP in various food matrices obtained 54 % to 85 % ZEN degradation. The findings in this study will promote the utilization of SHP as a cheap and renewable biocatalyst for degrading ZEN in food.

Abstract B052: Defining the lysosome proteome during tumor evolution

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a 5-year survival rate of 12%. PDAC tumors are highly reliant on nutrient scavenging pathways such as autophagy, and the lysosome – a degradative organelle which plays an essential role in the digestion and recycling of diverse cellular material. Lysosome and autophagy processes are regulated by the MiT/TFE family of transcription factors. In PDAC, MiT/TFE are uncoupled from normal regulatory mechanisms and have been shown to be constitutively nuclear, and therefore active. The function of lysosomes and the status of MiT/TFE over the course of tumor evolution and metastasis remains unknown. To fully understand the functions of the lysosome in cancer, our lab uses biochemical approaches to isolate intact lysosomes (termed LysoIP) from cultured PDAC cells, followed by mass spectrometry-based proteomics. Using this strategy, we have uncovered unique features and functions of PDAC lysosomes that promote cellular adaptation to stress in support of tumor growth. A limitation of lysosome isolation from 2D cultured cells is that it does not recapitulate lysosomal features associated with tumors growing in vivo. To address this limitation, we generated a genetically engineered mouse model of PDAC incorporating conditional expression of our ‘LysoTag’ (TMEM192-mRFP-3xHA) that enables capture and profiling of lysosomes at different stages of tumor evolution, including early stage, late stage, and metastatic disease. I have now established purification protocols for isolation of intact lysosomes with high efficiency and purity from both normal pancreas and diseased tissue. My preliminary analysis of pancreatic tumors, liver and lung metastases shows enrichment of bonafide resident lysosomal membrane proteins (Lamp1, Cln3, Npc1), and lumenal proteins (Ctsb, Lipa, Neu1), confirming successful isolation of intact lysosomes from these complex tissues. Further analysis highlights pathways and proteins that are unique to lysosomes of each tissue. For instance, lysosomes from the primary tumor show enrichment for pathways involved in ‘antigen processing and presentation of peptide antigen’ and ‘cellular response to oxidative stress’, while the liver and lung metastasis lysosomes show enrichment for ‘cholesterol metabolism’ and ‘mitochondrial gene expression’, respectively. Our analysis of lysosomes from the primary tumor, lung and liver metastasis highlights unique differences in the lysosome proteome of tumor cells growing in different tissues. Functional validation of our proteomics datasets will be needed to uncover novel roles for lysosomes in helping tumor cells adapt and grow in distinct tumor microenvironments. My ongoing studies will provide the first comprehensive atlas of lysosomal cargo and resident proteins that co-evolve with increasing tumor stage and help to inform novel therapeutic targets for treatment of PDAC. Citation Format: Grace A. Hernandez, Thuy Nguyen, Matthew Luy, Alexander Li, Longhui Qiu, Joseph D. Mancias, Rushika M. Perera. Defining the lysosome proteome during tumor evolution [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B052.

Sorbus spp. berries extraction in subcritical water: Bioactives recovery and antioxidant activity

With the aim of developing a sustainable and efficient extraction of polyphenols-rich fraction from Sorbus spp., Microwave-Assisted Subcritical Water Extraction (MASWE) was selected as the technique of choice. Although Sorbus spp. berries are rich in nutraceutical compounds, they are generally underutilised. The extracts obtained contain caffeoylquinic acids (i.e. chlorogenic acids), flavonoids, and organic acids, all of which are active components with potential applications in innovative functional foods due to their potent antioxidant activity. To optimise the extraction protocol, different temperatures and different species of the above genus were studied to improve the recovery of the polyphenolic fractions and obtain products with potential high bioactivity. The best resulting sample was studied more in detail using different colorimetric assays (total content of polyphenols, flavonoids and sugars – TPC, 406.43 mgGAE/gext; TFC, 13.5 mgQE/gext; TSC, 454.1 mgGlu/gext), purification streams, and LC-MS analyses. However, the most remarkable property observed is the outstanding antioxidant activity of the extract (4.74 mmolTrolox.eq/gext). In order to obtain a refined product rich in bioactive compounds, a purification protocol was performed leading to a final TPC selectivity of about 87 % and 10.12 mmolTrolox.eq/gext. Qualitative LC-MS analyses helped to identify chlorogenic acids as predominant in the extract, along with methyl esters of sorbic acid.

Affinity Purification of Anti-Parkin Antibodies from Rabbit Serum for their Application in Immunohistochemistry

Background:Parkin is a mitochondrial autophagy protein that is associated with Parkinson’s Disease (PD), as well as auditory function. Parkin dysfunction in the context of PD allows accumulation of protein aggregates, leading to dopaminergic neurotoxicity. Previous work has demonstrated that Parkin plays an essential role in normal auditory function, as Prkn-/- mice exhibit significantly reduced hearing sensitivity. However, Prkn-/- mice also experience a protective effect from cochlear hair cell death and hearing loss caused by aminoglycosides, which are commonly used antibiotics that can cause permanent hearing loss. Multiple antibodies marketed as suitable for immunohistochemistry (IHC) have been validated using Prkn-/- tissues; however, they exhibit non-specific activity. This project aims to generate a highly specific antibody for accurate and reliable detection of Parkin expression.
 Project Methods:Rabbits were immunized using two Parkin peptides, notated P1 and P2, and the resulting serum was collected. A two-step purification process was utilized in this project, first isolating the IgGs from serum via protein A/G columns, and secondly using affinity purification to obtain specific antibodies against antigens P1 and P2. Additionally, two different protocols for affinity purification were tested and compared. Purity of serum, IgG, and specific antibodies was assessed by Western blot and immunofluorescence.
 Results:Utilizing the first purification protocol, IHC antibodies were applied in Western blot, and all demonstrated a prominent ~50kD band specific to Parkin, with some degree of nonspecific binding. Immunostaining confirmed functionality of the IHC antibodies and revealed those against P2 exhibited higher binding specificity. The second purification protocol generated IHC antibodies of similar, if not slightly superior specific interaction.
 Conclusion/Implications:Further investigation of Parkin expression would facilitate a better understanding of how it may be associated with the development of PD, as well as auditory function, with the potential of utilizing Parkin as a therapeutic target in PD treatment and preventing aminoglycoside ototoxicity.

Fabrication of nanoporous multilayer graphene nanoplatelets membrane for water desalination

In recent years, graphene-based membranes are extensively explored for desalination process to fulfil the pure water scarcity. Still, there is a noticeable gap in the research on a technique that simultaneously deposits a membrane and also generates pores in the flake of membrane. In this work, we demonstrated a one-step and scalable protocol i.e. a conventional atmospheric plasma spraying (APS) to fabricate a graphene nanoplatelets (GNP) membrane for water desalination application. Various characterization tools: FE-SEM, Raman, and TEM etc. utilized to evaluate the deposition of GNPs flakes and induced sub-nanometer pores in the large area GNP membrane. Plasma-sprayed GNP membrane showed impressive desalination performance in terms of water flux, salt rejection, and permeability around ~67 Lm−2 h−1, ~99 %, and ~ 115 Lm−2h−1bar−1 respectively at 0.6 bar of transmembrane pressure (TMP). This performance of the GNP membrane was attributed to the critical role of graphene in terms of its structures, including induced pores within graphene sheets and its functions. Our deposited GNP membrane is cost-effective and showed the applicability of conventional plasma spraying in the design of nanomaterials-based membranes for various water purification protocols.

Crystallographic and biochemical analyses of a far-red allophycocyanin to address the mechanism of the super-red-shift.

Far-red absorbing allophycocyanins (APC), identified in cyanobacteria capable of FRL photoacclimation (FaRLiP) and low-light photoacclimation (LoLiP), absorb far-red light, functioning in energy transfer as light-harvesting proteins. We report an optimized method to obtain high purity far-red absorbing allophycocyanin B, AP-B2, of Chroococcidiopsis thermalis sp. PCC7203 by synthesis in Escherichia coli and an improved purification protocol. The crystal structure of the trimer, (PCB-ApcD5/PCB-ApcB2)3, has been resolved to 2.8Å. The main difference to conventional APCs absorbing in the 650-670nm range is a largely flat chromophore with the co-planarity extending, in particular, from rings BCD to ring A. This effectively extends the conjugation system of PCB and contributes to the super-red-shifted absorption of the α-subunit (λmax = 697nm). On complexation with the β-subunit, it is even further red-shifted (λmax, absorption = 707nm, λmax, emission = 721nm). The relevance of ring A for this shift is supported by mutagenesis data. A variant of the α-subunit, I123M, has been generated that shows an intense FR-band already in the absence of the β-subunit, a possible model is discussed. Two additional mechanisms are known to red-shift the chromophore spectrum: lactam-lactim tautomerism and deprotonation of the chromophore that both mechanisms appear inconsistent with our data, leaving this question unresolved.

Purification for Hybrid Entanglement between Discrete‐ and Continuous‐Variable States

AbstractHybrid entangled states (HES) have attracted significant attention and been utilized in various quantum information processing applications. However, like many other entangled states, maximally entangled HES may degrade to mixed states due to environmental noise and operational imperfections. In this paper, a hybrid entanglement purification protocol (HEPP) for the HES, which consists of photon‐number state and coherent state is proposed. This HEPP is designed to effectively purify a bit‐flip error occurring in any qubit of the HES. Furthermore, HEPP is extended to a general condition, say, the multi‐party scenario, and integrates the generation of HES into the HEPP. Moreover, if different initial mixed states are chosen, then the residual entanglement can be utilized to distill high‐quality entanglement. The HEPP has important applications in the future quantum information processing field.

An optimized purification protocol for enzymatically synthesized S-adenosyl-L-methionine (SAM) for applications in solution state infrared spectroscopic studies

S-adenosyl-L-methionine (SAM) is an abundant biomolecule used by methyltransferases to regulate a wide range of essential cellular processes such as gene expression, cell signaling, protein functions, and metabolism. Despite considerable effort, there remain many specificity challenges associated with designing small molecule inhibitors for methyltransferases, most of which exhibit off-target effects. Interestingly, NMR evidence suggests that SAM undergoes conformeric exchange between several states when free in solution. Infrared spectroscopy can detect different conformers of molecules if present in appreciable populations. When SAM is noncovalently bound within enzyme active sites, the nature and the number of different conformations of the molecule are likely to be altered from when it is free in solution. If there are unique structures or different numbers of conformers between different methyltransferase active sites, solution-state information may provide promising structural leads to increase inhibitor specificity for a particular methyltransferase. Toward this goal, frequencies measured in SAM’s infrared spectra must be assigned to the motions of specific atoms via isotope incorporation at discrete positions. The incorporation of isotopes into SAM’s structure can be accomplished via an established enzymatic synthesis using isotopically labeled precursors. However, published protocols produced an intense and highly variable IR signal which overlapped with many of the signals from SAM rendering comparison between isotopes challenging. We observed this intense absorption to be from co-purifying salts and the SAM counterion, producing a strong, broad signal at 1100 cm−1. Here, we report a revised SAM purification protocol that mitigates the contaminating salts and present the first IR spectra of isotopically labeled CD3-SAM. These results provide a foundation for isotopic labeling experiments of SAM that will define which atoms participate in individual molecular vibrations, as a means to detect specific molecular conformations.