Affinity Chromatography Of Proteins Research Articles

A Spy Chemistry-Based Method for Purification of Proteins with Authentic N-Termini

Protein purification is essential in life sciences and biomanufacturing. Tag-mediated protein affinity chromatography (AC) enables the preparation of recombinant proteins with medium to high purity. However, traditional AC methods often require expensive resins and additional tag removal steps. Here, we introduce a purification method for proteins with authentic N-termini based on reusable SpyDock-modified epoxy resin and a pH-inducible self-cleavage intein. This method was validated using SpyTag002-fused red fluorescent protein (RFP) and applied to purify three model proteins: glutathione S-transferase (GST), human growth hormone (hGH), and the nanobody caplacizumab, directly from cell lysates. The purified proteins achieved high purities (92–98%) and comparable yields to the commercial His-tag method. The preparation of the SpyDock-modified resin is straightforward, and SpyDock can be easily produced via standard Escherichia coli fermentation processes, making it potentially suitable for industrial-scale applications.

The efficient synthesis and purification of 2'3'- cGAMP from Escherichia coli.

Agonists of the stimulator of interferon genes (STING) pathway are being explored as potential immunotherapeutics for the treatment of cancer and as vaccine adjuvants for infectious diseases. Although chemical synthesis of 2'3' - cyclic Guanosine Monophosphate-Adenosine Monophosphate (cGAMP) is commercially feasible, the process results in low yields and utilizes organic solvents. To pursue an efficient and environmentally friendly process for the production of cGAMP, we focused on the recombinant production of cGAMP via a whole-cell biocatalysis platform utilizing the murine cyclic Guanosine monophosphate-Adenosine monophosphate synthase (mcGAS). In E. coli BL21(DE3) cells, recombinant expression of mcGAS, a DNA-dependent enzyme, led to the secretion of cGAMP to the supernatants. By evaluating the: (1) media composition, (2) supplementation of divalent cations, (3) temperature of protein expression, and (4) amino acid substitutions pertaining to DNA binding; we showed that the maximum yield of cGAMP in the supernatants was improved by 30% from 146 mg/L to 186 ± 7 mg/mL under optimized conditions. To simplify the downstream processing, we developed and validated a single-step purification process for cGAMP using anion exchange chromatography. The method does not require protein affinity chromatography and it achieved a yield of 60 ± 2 mg/L cGAMP, with <20 EU/mL (<0.3 EU/μg) of endotoxin. Unlike chemical synthesis, our method provides a route for the recombinant production of cGAMP without the need for organic solvents and supports the goal of moving toward shorter, more sustainable, and more environmentally friendly processes.

Learning to Read and Write in the Language of Proteins

Learning to Read and Write in the Language of Proteins

The His-tag as a decoy modulating preferred orientation in cryoEM.

The His-tag is a widely used affinity tag that facilitates purification by means of affinity chromatography of recombinant proteins for functional and structural studies. We show here that His-tag presence affects how coproheme decarboxylase interacts with the air-water interface during grid preparation for cryoEM. Depending on His-tag presence or absence, we observe significant changes in patterns of preferred orientation. Our analysis of particle orientations suggests that His-tag presence can mask the hydrophobic and hydrophilic patches on a protein's surface that mediate the interactions with the air-water interface, while the hydrophobic linker between a His-tag and the coding sequence of the protein may enhance other interactions with the air-water interface. Our observations suggest that tagging, including rational design of the linkers between an affinity tag and a protein of interest, offer a promising approach to modulating interactions with the air-water interface.

Investigating a putative transcriptional regulatory protein encoded by Rv1719 gene of Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the causative agent of tuberculosis, demonstrates immense plasticity with which it adapts to a highly dynamic and hostile host environment. This is facilitated by a web of signalling pathways constantly modulated by a multitude of proteins that regulate the flow of genetic information inside the pathogen. Transcription factors (TFs) belongs to one such family of proteins that modulate the signalling by regulating the abundance of proteins at the transcript level. In the current study, we have characterized the putative transcriptional regulatory protein encoded by the Rv1719 gene of Mycobacterium tuberculosis. This TF belongs to the IclR family of proteins with orthologs found in both bacterial and archaeal species. We cloned the Rv1719 gene into the pET28a expression vector and performed heterologous expression of the recombinant protein with E coli as the host. Further, optimization of the purification protocol by affinity chromatography and characterization of proteins for their functional viability has been demonstrated using various biochemical and/or biophysical approaches. Scale-up of purification yielded approximately 30mg of ~ 28kDa protein per litre of culture. In-silico protein domain analysis of Rv1719 protein predicted the presence of the helix-turn-helix (HTH) domain suggesting its ability to bind DNA sequence and modulate transcription; a hallmark of a transcriptional regulatory protein. Further, by performing electrophoretic mobility shift assay (EMSA) we demonstrated that the protein binds to a specific DNA fragment harboring the probable binding site of one of the predicted promoters.

In situ synthesis and unidirectional insertion of membrane proteins in liposome-immobilized silica stationary phase for rapid preparation of microaffinity chromatography

Cell membrane affinity chromatography has been widely applied in membrane protein (MP)-targeted drug screening and interaction analysis. However, in current methods, the MP sources are derived from cell lines or recombinant protein expression, which are time-consuming for cell culture or purification, and also difficult to ensure the purity and consistent orientation of MPs in the chromatographic stationary phase. In this study, a novel in situ synthesis membrane protein affinity chromatography (iSMAC) method was developed utilizing cell-free protein expression (CFE) and covalent immobilized affinity chromatography, which achieved efficient in situ synthesis and unidirectional insertion of MPs into liposomes in the stationary phase. The advantages of iSMAC are: 1) There is no need to culture cells or prepare recombinant proteins; 2) Specific and purified MPs with stable and controllable content can be obtained within 2 h; 3) MPs maintain the transmembrane structure and a consistent orientation in the chromatographic stationary phase; 4) The flexible and personalized construction of cDNAs makes it possible to analyze drug binding sites. iSMAC was successfully applied to screen PDGFRβ inhibitors from Salvia miltiorrhiza and Schisandra chinensis. Micro columns prepared by in-situ synthesis maintain satisfactory analysis activity within 72 h. Two new PDGFRβ inhibitors, salvianolic acid B and gomisin D, were screened out with KD values of 13.44 and 7.39 μmol/L, respectively. In vitro experiments confirmed that the two compounds decreased α-SMA and collagen Ӏ mRNA levels raised by TGF-β in HSC-T6 cells through regulating the phosphorylation of p38, AKT and ERK. In vivo, Sal B could also attenuate CCl4-induced liver fibrosis by downregulating PDGFRβ downstream related protein levels. The iSMAC method can be applied to other general MPs, and provides a practical approach for the rapid preparation of MP-immobilized or other biological solid-phase materials.

Protease inhibitor activity of plant natural products as leishmanicine agents / Atividade de inibidores de proteases de produtos naturais contra agentes leishmanicidas

Objective: Investigate plant natural products with inhibitory activity of Leishmania proteases, because the inhibition of certain proteases induce the parasite death. Methods: Descriptive/exploratory study, integrative review type. The search was performed in the following databases: Scientific Electronic Library OnLine (SciELO), Medical Literature Analysis and Retrieval System Online (MEDLINE), Latin American and Caribbean Health Science Literature Database (LILACS) and PUBMED. The descriptors were used in combination, from the consultation in the Descriptors in Health Sciences (DECS) and Medical Subject Headings (MeSH), were: (Leishmania) AND (Protease Inhibitor) AND (Vegetable extracts); (Leishmania) AND (Protease Inhibitor) AND (Plant Extracts). Studies in English and Portuguese, published between 2000 and 2020, were included. Finally, the articles were categorized and analyzed. Results and discussion: This review included eight studies, published between 2000 and 2020. There were two publications in 2014, 2017 and 2019, in relation to the language, all eight studies were published in English. Five studies were carried out in India, two in Brazil and one in Iran. Seven of these were found in PUBMED database and one from MEDLINE, all were experimental, comprising in vitro research or using mice as animal models. Conclusions: It was noted that they use similar protocols for the isolation of protease inhibitor from plant species (affinity chromatography) and proteases from Leishmania sp. (cell lysis and protein precipitation and affinity chromatography). However, research involving Leishmania protease inhibitors are still scarce, requiring further studies on the subject, since the understanding of the functioning of these molecules has much to contribute to the development of new therapeutic targets.

Encapsulating IM7-Displaying Yeast Cells in Calcium Alginate Beads for One-Step Protein Purification and Multienzyme Biocatalysis.

There are several commercial chromatographic systems for protein purification; however, development of cost-effective 3H-grade (high yield, high purity, and high activity) purification approaches is highly demanded. Here, we establish a methodology for encapsulating the IM7-displaying yeast cells in calcium alginate beads. Taking advantage of this biomaterial-based affinity chromatography, rapid and cost-effective purification of proteins with over 90% purity in a single step is achieved. Moreover, our system enables coating the multienzyme complex to produce reusable immobilized cells for efficient cascade biotransformation. Together, the present method has great application potentials not only in the laboratory but also in the industry for production of protein products as well as biocatalysis.

Nickel Ferrite Nanoparticles as an Adsorbent for Immobilized Metal Affinity Chromatography of Proteins

A simple method of preparing amorphous nickel ferrite nanoparticles of about 5nm diameter is described. These particles were characterized by dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM) and selected area electron diffraction (SAED). The nanoparticles were evaluated for their use as a magnetic material for immobilized metal affinity chromatography (IMAC). The ferrite nanoparticles bound to bovine serum albumin (BSA) and the binding fitted Langmuir isotherm model. A high capacity of 916mg BSA/g dried nanoparticle was observed. Six proteins (Soybean trypsin inhibitor (STI), lactate dehydrogenase (LDH), papain, catalase, β-galactosidase and casein) were used and all were found to bind at >90% level (except papain which showed 84% binding). All the proteins except LDH and β-galactosidase could be eluted with 1M imidazole and with % activity recovery of >80%. Papain could be purified from its dried crude latex by 5-fold and purified papain showed a single band on SDS-PAGE. These nanoparticles constitute a high capacity and are magnetic material useful for IMAC and do not require any pre-functionalization.

Current Trends in Protein Purification : A Review

The present review is based on papers published between 1990 and 2020 and gives Comparative information about the most common protein purification techniques Gel-Filtration, Chromatography, Ion-Exchange Chromatography, Electrophoresis, Affinity Chromatography, and Dialysis, High-Pressure Liquid Chromatography. and their applications.

IgGs-Abzymes from the Sera of Patients with Systemic Lupus Erythematosus Hydrolyzed miRNAs.

Background and ObjectivesSystemic lupus erythematosus (SLE) is an inflammatory disease. The sera of SLE patients contain antibodies-abzymes hydrolyzing myelin basic protein (MBP), DNA, nucleotides, and oligosaccharides. The blood of SLE patients contains an increased amount of some specific miRNAs. This study aimed to analyze a possible hydrolysis of eight microRNAs found in the blood of SLE patients with high frequency by blood antibodies-abzymes.Patients and MethodsUsing affinity chromatography of the serum proteins of SLE patients and healthy donors on protein G-Sepharose and following FPLC gel filtration, electrophoretically homogeneous IgG preparations containing no impurities of canonical RNases were obtained. These preparations were used to analyze their activity in the hydrolysis of eight miRNAs.ResultsIt was shown that SLE IgGs hydrolyze very efficiently four neuroregulatory miRNAs (miR-219-2-3p, miR-137, miR-219a-5p, and miR-9-5p) and four immunoregulatory miRNAs (miR-326, miR-21-3p, miR-155-5p, and miR-146a-3p). To demonstrate that the miRNAs hydrolysis is an intrinsic property of SLE IgGs, several rigid criteria were checked. Only some IgGs of healthy donors showed very weak, but reliably detectable activity in the hydrolysis miRNAs. The average activity of SLE patients IgGs according to median values is statistically significant 84.8-fold higher than that of healthy donors. The maximum and comparable average activity (RA) was observed in the hydrolysis of three miRAs: miR-9-5p, miR-155-5p, and miR-326. MiR-9-5p plays an important role in the development of lupus nephritis, while miR-326 activates the production of antibodies by B cells. The major and moderate specific sites of the hydrolysis of each miRNA were revealed. The hydrolysis of eight microRNAs was mostly site specific. Several SLE IgGs hydrolyzed some miRNAs demonstrating a combination of site-specific and non-specific splitting.ConclusionSince inflammatory processes in SLE are associated with the change in miRNAs expression, the decrease in their concentration due to hydrolysis by autoantibodies-abzymes may be important for SLE pathogenesis.

Differential secretome of pancreatic cancer cells in serum-containing conditioned medium reveals CCT8 as a new biomarker of pancreatic cancer invasion and metastasis

BackgroundPancreatic cancer is a malignancy with a very poor prognosis. The emergence of liquid biopsy is expected to achieve accurate early diagnosis through detection of tumor-derived secreted proteins in the blood. Early diagnosis and treatment of pancreatic cancer could help to improve prognosis.MethodsThe pretreatment approach of samples can have a major effect on downstream analysis. In this study, we used a pair of homologous pancreatic cancer cell supernatants with different capacities for invasion and metastasis to examine secreted proteins in the conditioned media without the removal of fetal bovine serum, namely through size exclusion chromatography combined with high-abundance protein affinity chromatography to enrich low-concentration protein, followed by mass spectrometry using triple dimethyl labeling. Identification of proteins was performed using an online public database and western blot.ResultsMass spectrometry data revealed 77 proteins with quantitative properties, of which 12 proteins had over a 1.5-fold difference (in the supernatant of the highly invasive pancreatic cancer cell line PC-1.0, the expression of 8 proteins were increased and the expression of 4 proteins were decreased). Bioinformatics analysis results showed that CCT8, CTSL, SAA1, IGF2 are secreted via the exosome pathway. According to the literature, with the exception of CCT8, the other three proteins can be detected in blood samples of pancreatic cancer patients, and they can be used as prognostic markers. Western blot results were used to validate consistency with MS results.ConclusionThis study found that CCT8 can be used as a liquid biopsy marker to assess the prognosis of pancreatic cancer patients.

Chikungunya virus requires cellular chloride channels for efficient genome replication.

Chikungunya virus (CHIKV) is a re-emerging, pathogenic alphavirus that is transmitted to humans by Aedes spp. mosquitoes—causing fever and debilitating joint pain, with frequent long-term health implications and high morbidity. The CHIKV lifecycle is poorly understood and specific antiviral therapeutics or vaccines are lacking. In this study, we investigated the role of host-cell chloride (Cl-) channels on CHIKV replication.We demonstrate that specific pharmacological Cl- channel inhibitors significantly inhibit CHIKV replication in a dose-dependent manner, suggesting that Cl-channels are pro-viral factors in human cells. Further analysis of the effect of the inhibitors on CHIKV attachment, entry, viral protein expression and replicon replication demonstrated that Cl- channels are specifically required for efficient CHIKV genome replication. This was conserved in mosquito cells, where CHIKV replication and genome copy number was significantly reduced following Cl- channel inhibition. siRNA silencing identified chloride intracellular channels 1 and 4 (CLIC1 and CLIC4, respectively) as required for efficient CHIKV replication and protein affinity chromatography showed low levels of CLIC1 in complex with CHIKV nsP3, an essential component of the viral replication machinery. In summary, for the first time we demonstrate that efficient replication of the CHIKV genome depends on cellular Cl- channels, in both human and mosquito cells and identifies CLIC1 and CLIC4 as agonists of CHIKV replication in human cells. We observe a modest interaction, either direct or indirect, between CLIC1 and nsP3 and hypothesize that CLIC1 may play a role in the formation/maintenance of CHIKV replication complexes. These findings advance our molecular understanding of CHIKV replication and identify potential druggable targets for the treatment and prevention of CHIKV mediated disease.

Isolation and characterization of acute phase protein C-reactive protein in shark serum: a potential biomarker for assessing health in wild sharks

Abstract The acute phase protein, C-reactive protein (CRP), has become an established clinical biomarker in both animal and human health, where it is used to assess the degree of acute and/or chronic inflammation. CRP has also been proposed as a potential biomarker for measuring health in sharks by indicating when a shark is experiencing an inflammatory state or is under a chronic state of stress that may have significant physiological impact. Despite the promise in using increased levels of CRP as well as other acute phase proteins as indicators of compromised health, there has not to date been a developed shark-specific assay to accurately measure CRP. An initial step in the development of a specific bioassay for CRP is isolation and characterization of the protein. The purpose of the present study is to use both field and laboratory techniques to obtain serum samples and isolate CRP from nurse sharks, Ginglymostoma cirratum. A secondary aim is to characterize the molecule and produce shark CRP specific antisera. A two step isolation procedure is now being refined to positively identify the pentraxins present and characterize them further. Protein affinity chromatography was used to identify serum amyloid P component (SAP) in addition to CRP given that they are both pentraxin proteins displaying similar binding properties. Isolated protein bands resulting from concentrated chromatographic peaks were analyzed by LC-MS/MS. Results thus far include the isolation of a pentraxin domain-containing protein within shark serum.

Preparation and identification of polyclonal antibody against human cytomegalovirus encoding protein UL23

Human cytomegalovirus (HCMV), a member of the human herpesvirus family, is a common opportunistic virus causing severe ailments and deaths in people with immature or compromised immune systems. UL23 is a virion protein found in the tegument and is expressed in the cytoplasm in HCMV infected cells. However, UL23 is dispensable for viral replication in cultured cells and little is currently known about its function. In order to further study of UL23, polyclonal antibody of UL23 was prepared. UL23 gene fragment was cloned from HCMV Towne by PCR and ligated into pET28a (+). The recombinant plasmid pET28a (+)-UL23 was transformed into E.coli BL21(DE3) to induce expression of the target protein. Then we efficiently purified the recombinant protein affinity chromatography under unique denaturation conditions. Recombinant UL23 protein was used as immunogen to inoculate New Zealand white rabbits and the sera was collected after the fourth immunization. UL23 Polyclonal antibody was purified from antisera using CNBr-activated Sepharose 4 beads. Our UL23 Polyclonal antibody showed specific reaction with UL23 in ELISA, Western-blot and immunofluorescence. More importantly, UL23 Polyclonal antibody could specifically recognize UL23 protein in HCMV infected cells, which laid a foundation for further study of HCMV UL23.

Development of an indirect ELISA assay to evaluation of the adaptive immune response of pacu (Piaractus mesopotamicus).

The pacu is one of the most important species for Brazilian fish farming and is considered emerging in the global aquaculture. Despite its importance, no effective tool for evaluation of the adaptive immune response of this species has been developed. Therefore, this study aimed the development and standardization of indirect ELISA for the measurement of pacu antigen-specific antibodies using polyclonal rabbit anti-pacu IgM used as detector antibody. For this purpose was isolated and purificated of pacu IgM using mannose-binding protein affinity chromatography and produced specific polyclonal antibodies against heavy and light chains pacu IgM, that showed a molecular weight of 72 kDa and 26 kDa, respectively. Polyclonal antibodies obtained demonstrated specificity with heavy and light Ig chains of pacu serum in western blotting. These polyclonal antibodies allowed the development of an indirect ELISA assay of high sensitivity and specificity for the detection and quantification of pacu IgM antibodies immunized with bovine IgG. In conclusion, this approach has great potential to improve the monitoring of vaccine-induced immune responses and help develop immunodiagnostic and epidemiological studies of infectious diseases in pacu systems.

P.1.017 - New link between inflammation and schizophrenia: IgG with nuclease activity

Introduction Schizophrenia is known to be associated with the disorganization of neurotransmission processes in the brain as well as immunological disturbances [1]. Inflammation in the perinatal period can lead to a violation of synaptic plasticity and pruning of synapses in the adulthood [2]. Also, microRNAs play an important role in neuronal development. The microRNA expression profiles in the circulation and brain are altered in schizophrenia. It is shown that a change in the levels of microRNAs in the blood serum or brain leads to a violation of behavior [3]. We have shown that the formation of catalytic antibodies (abzymes) is one of the signs of immune system pathology. Catalytic antibodies hydrolyzing DNA were detected in the serum of patients with schizophrenia [4]. We assume that antibodies hydrolyzing the circulating regulatory nucleic acids can play a role in schizophrenia pathogenesis. Objectives To investigate a) microRNA-hydrolyzing activity of IgG antibodies from the serum of patients with schizophrenia; b) correlation of the microRNA-hydrolyzing activity level with clinical features of the disease; c) the cytotoxicity of isolated IgG on the human neuroblastoma cell culture IMR-32. Materials and Methods In our study, 35 patients with a verified diagnosis of simple or paranoid schizophrenia and 20 healthy donors were included. The average age in the group of patients was 35.8±8.9 years, duration of disease – 9.1±5.4 years. Electrophoretically and immunologically homogeneous IgGs were obtained by affinity chromatography of the serum proteins under conditions removing non-specifically bound proteins. The microRNA-hydrolyzing activity of IgG was detected by the level of hydrolysis of two neuro-specific fluorescently labeled microRNAs (miR-137 and miR-219a-2-3p). The products of the hydrolysis were analyzed by PAGE under denaturing conditions using laser scanner. Cytotoxicity of antibodies on human neuroblastoma cell culture IMR-32 was studied using MTT-test. Statistical analysis was carried out using the Pearson’s correlation in the Statistica 10 software. Results We have shown for the first time that IgG from all schizophrenia patients possesses microRNA-hydrolyzing activity. Using strict criteria, we proved that the observed activity is an intrinsic property of IgGs in schizophrenia. Interestingly, the microRNA-hydrolyzing activity level on miR-137 is strongly negatively correlated with the disease duration (R=–0.879). The activity decreased with increasing duration of the disease. Also, the level of RNase activity on miR-219a-2-3p was positively correlated with the values of the General Psychopathology PANSS scale and the PANSS Total score (R>0.8). It was shown that the RNase activity is metal-dependent. The kinetic parameters of nuclease reactions catalyzed by IgG were significantly lower than that for native enzymes. Besides, we detected the major hydrolysis sites of two neuro-specific microRNAs. Also, we show that IgG from schizophrenia patients with nuclease activity reduced the survival rate neuroblastoma IMR-32 cell culture by 10-28%. Conclusion We propose that immunological disorders can play an important role in schizophrenia, especially in the prodromal period. The environment and genetic predisposition can lead to inflammation and the formation of antibodies hydrolyzing the microRNAs. These antibodies can contribute to changes in the level of circulating microRNAs and schizophrenia development.

Molecular characterization of Babesia microti thioredoxin (BmTrx2) and its expression patterns induced by antiprotozoal drugs

BackgroundHuman babesiosis is an infectious disease that is epidemic in various regions all over the world. The predominant causative pathogen of this disease is the intra-erythrocytic parasite Babesia microti. The thioredoxin system is one of the major weapons that is used in the resistance to the reactive oxygen species (ROS) and reactive nitrogen species (RNS) produced by host immune system. In other intra-erythrocytic apicomplexans like the malaria parasite Plasmodium falciparum, anti-oxidative proteins are promising targets for the development of anti-parasitic drugs. However, to date, the sequences and biological properties of thioredoxins and thioredoxin-like molecules of B. microti remain unknown. Understanding the molecular characterization and function of B. microti thioredoxins may help to develop anti-Babesia drugs and controlling babesiosis.MethodsThe full-length B. microti thioredoxin 2 (BmTrx2) gene was obtained using a rapid amplification of cDNA ends (RACE) method, and the deduced BmTrx2 amino acid sequence was analyzed using regular bioinformatics tools. Recombinant BmTrx2 protein was expressed in vitro and purified using His-tag protein affinity chromatography resins. Reverse transcription PCR, quantitative real-time PCR and Western blot were employed to detect the expression and native proteins of BmTrx2. Indirect immunofluorescence assay was used to localize BmTrx2 in B. microti. Bovine insulin reduction assays were used to determine the enzyme activity of the purified recombinant BmTrx2 protein.ResultsThe full-length BmTrx2 was 564 bp with a 408 bp open reading frame encoding a protein of 135 amino acids. The predicted molecular weight of the protein was 15.5 kDa. A conserved thioredoxin-like family domain was found in BmTrx2. The expression of BmTrx2 was upregulated on both the third and eighth day post-infection in mice, whereas expression was downregulated during the beginning and later stages. The results of Western blot analysis showed the native BmTrx2 in parasite lysates could be detected by mouse anti-BmTrx2 serum and that the recombinant BmTrx2 protein could be recognized by serum of B. microti-infected mice. Immunofluorescence microscopy showed that BmTrx2 localized in the cell cytoplasm of B. microti merozoites in B. microti-infected red blood cells. The results of bovine insulin reduction assay indicated the purified recombinant BmTrx2 protein possesses antioxidant enzyme activity. Dihydroartemisinin and quinine, known anti-malaria drugs, and clindamycin, a known anti-babesiosis drug, induced significantly higher upregulation of BmTrx2 mRNA.ConclusionsOur results indicate that BmTrx2 is a functional enzyme with antioxidant activity and may be involved in the response of B. microti to anti-parasite drugs.

Sequence-Specific Affinity Chromatography of Bacterial Small Regulatory RNA-Binding Proteins from Bacterial Cells.

Bacterial small RNA molecules (sRNAs) are increasingly recognized as central regulators of bacterial stress responses and pathogenesis. In many cases, RNA-binding proteins are critical for the stability and function of sRNAs. Previous studies have adopted strategies to genetically tag an sRNA of interest, allowing isolation of RNA-protein complexes from cells. Here we present a sequence-specific affinity purification protocol that requires no prior genetic manipulation of bacterial cells, allowing isolation of RNA-binding proteins bound to native RNA molecules.

Immobilized fusion protein affinity chromatography combined with HPLC–ESI-Q-TOF-MS/MS for rapid screening of PPARγ ligands from natural products

Screening agonists of peroxisome proliferator-activated receptor-γ (PPARγ) from natural products is particularly motivated by the need for effective anti-diabetic agents. However, method for direct identification of PPARγ ligands from a complex sample is rarely reported. Here we propose a novel immobilized fusion protein affinity chromatography (IFPAC) to achieve rapid multicomponent screening. First, functional human PPARγ ligand binding domain (hPPARγLBD) was bacterially produced by fusion to glutathione S-transferase (GST). The unpurified GST-hPPARγLBD was directly applied to a 96-well filter plate prepacked with glutathione sepharose. Due to the strong affinity between GST and glutathione, the fusion protein could selectively attach to the glutathione matrix with an oriented immobilization, which was rapidly purified under non-denaturing conditions. Experimental results indicated that the prepared 96-affinity column array exhibited excellent selectivity and sensitivity for fishing novel interacting compounds. The proposed approach was applied in the high-throughput screening of PPARγ ligands from natural products, followed by rapid characterization of active compounds using HPLC–ESI-Q-TOF-MS/MS. Isochlorogenic acid A in Dendranthema indicum flowers was found to be a PPARγ ligand. Our findings suggested the IFPAC could be a powerful tool for drug discovery from natural products.