A novel approach for separating bacteriophages from other bacteriophages using affinity chromatography and phage display.
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ABSTRACT:
SUBMITTER: Ceglarek I
PROVIDER: S-EPMC3827602 | biostudies-other | 2013
REPOSITORIES: biostudies-other
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Project description:BACKGROUND: Affinity chromatography is one of the most efficient protein purification strategies. This technique comprises a one-step procedure with a purification level in the order of several thousand-fold, adaptable for various proteins, differentiated in their size, shape, charge, and other properties. The aim of this work was to verify the possibility of applying affinity chromatography in bacteriophage purification, with the perspective of therapeutic purposes. T4 is a large, icosahedral phage that may serve as an efficient display platform for foreign peptides or proteins. Here we propose a new method of T4 phage purification by affinity chromatography after its modification with affinity tags (GST and Histag) by in vivo phage display. As any permanent introduction of extraneous DNA into a phage genome is strongly unfavourable for medical purposes, integration of foreign motifs with the phage genome was not applied. The phage was propagated in bacteria expressing fusions of the phage protein Hoc with affinity tags from bacterial plasmids, independently from the phage expression system. RESULTS: Elution profiles of phages modified with the specific affinity motifs (compared to non-specific phages) document their binding to the affinity resins and effective elution with standard competitive agents. Non-specific binding was also observed, but was 102-105 times weaker than the specific one. GST-modified bacteriophages were also effectively released from glutathione Sepharose by proteolytic cleavage. The possibility of proteolytic release was designed at the stage of expression vector construction. Decrease in LPS content in phage preparations was dependent on the washing intensity; intensive washing resulted in preparations of 11-40 EU/ml. CONCLUSIONS: Affinity tags can be successfully incorporated into the T4 phage capsid by the in vivo phage display technique and they strongly elevate bacteriophage affinity to a specific resin. Affinity chromatography can be considered as a new phage purification method, appropriate for further investigations and development.
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Project description:Although the etiology of multiple sclerosis is not yet understood, it is accepted that its pathogenesis involves both autoimmune and neurodegenerative processes, in which the role of autoreactive T-cells has been elucidated. Instead, the contribution of humoral response is still unclear, even if the presence of intrathecal antibodies and B-cells follicle-like structures in meninges of patients has been demonstrated. Several myelin and non-myelin antigens have been identified, but none has been validated as humoral biomarker. In particular autoantibodies against myelin proteins have been found also in healthy individuals, whereas non-myelin antigens have been implicated in neurodegenerative phase of the disease. To provide further putative autoantigens of multiple sclerosis, we investigated the antigen specificity of immunoglobulins present both in sera and in cerebrospinal fluid of patients using phage display technology in a new improved format. A human brain cDNA phage display library was constructed and enriched for open-read-frame fragments. This library was selected against pooled and purified immunoglobulins from cerebrospinal fluid and sera of multiple sclerosis patients. The antigen library was also screened against an antibody scFv library obtained from RNA of B cells purified from the cerebrospinal fluid of two relapsing remitting patients. From all biopanning a complex of 14 antigens were identified; in particular, one of these antigens, corresponding to DDX24 protein, was present in all selections. The ability of more frequently isolated antigens to discriminate between sera from patients with multiple sclerosis or other neurological diseases was investigated. The more promising novel candidate autoantigens were DDX24 and TCERG1. Both are implicated in RNA modification and regulation which can be altered in neurodegenerative processes. Therefore, we propose that they could be a marker of a particular disease activity state.
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Project description:Molecular evolution is an important step in the development of therapeutic antibodies. However, the current method of affinity maturation is overly costly and labor-intensive because of the repetitive mutation experiments needed to adequately explore sequence space. Here, we employed a long short term memory network (LSTM)-a widely used deep generative model-based sequence generation and prioritization procedure to efficiently discover antibody sequences with higher affinity. We applied our method to the affinity maturation of antibodies against kynurenine, which is a metabolite related to the niacin synthesis pathway. Kynurenine binding sequences were enriched through phage display panning using a kynurenine-binding oriented human synthetic Fab library. We defined binding antibodies using a sequence repertoire from the NGS data to train the LSTM model. We confirmed that likelihood of generated sequences from a trained LSTM correlated well with binding affinity. The affinity of generated sequences are over 1800-fold higher than that of the parental clone. Moreover, compared to frequency based screening using the same dataset, our machine learning approach generated sequences with greater affinity.
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Project description:ABTAG is a camelid single-domain antibody (sdAb) that binds to bovine serum albumin (BSA) with low picomolar affinity. In surface plasmon resonance (SPR) analyses using BSA surfaces, bound ABTAG can be completely dissociated from the BSA surfaces at low pH, over multiple cycles, without any reduction in the capacity of the BSA surfaces to bind ABTAG. A moderate throughput, SPR-based, antibody screening assay exploiting the unique features of ABTAG is described. Anti-carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) sdAbs were isolated from a phage-displayed sdAb library derived from the heavy chain antibody repertoire of a llama immunized with CEACAM6. Following one or two rounds of panning, enriched clones were expressed as ABTAG fusions in microtiter plate cultures. The sdAb-ABTAG fusions from culture supernatants were captured on BSA surfaces and CEACAM6 antigen was then bound to the captured molecules. The SPR screening method gives a read-out of relative expression levels of the fusion proteins and kinetic and affinity constants for CEACAM6 binding by the captured molecules. The library was also panned and screened by conventional methods and positive clones were subcloned and expressed for SPR analysis. Compared to conventional panning and screening, the SPR-based ABTAG method yielded a considerably higher diversity of binders, some with affinities that were three orders of magnitude higher affinity than those identified by conventional panning.
| S-EPMC5674936 | biostudies-literature
Project description:The latest coronavirus disease outbreak, COVID-19, has brought attention to viral infections which have posed serious health threats to humankind throughout history. The rapid global spread of COVID-19 is attributed to the increased human mobility of today's world, yet the threat of viral infections to global public health is expected to increase continuously in part due to increasing human-animal interface. Development of antiviral agents is crucial to combat both existing and novel viral infections. Recently, there is a growing interest in peptide/protein-based drug molecules. Antibodies are becoming especially predominant in the drug market. Indeed, in a remarkably short period, four antibody therapeutics were authorized for emergency use in COVID-19 treatment in the US, Russia, and India as of November 2020. Phage display has been one of the most widely used screening methods for peptide/antibody drug discovery. Several phage display-derived biologics are already in the market, and the expiration of intellectual property rights of phage-display antibody discovery platforms suggests an increment in antibody drugs in the near future. This review summarizes the most common phage display libraries used in antiviral discovery, highlights the approaches employed to enhance the antiviral potency of selected peptides/antibody fragments, and finally provides a discussion about the present status of the developed antivirals in clinic.
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Project description:BACKGROUND:The transmembrane glycoprotein CD133 is believed to be a marker of adult prostate stem cells and cancer stem/initiating cells. Investigating the role of CD133 in the normal biology of the prostate and in cancer is complicated by the lack of a sensitive and accurate antibody for its detection. Here, we describe the characterization of a unique antibody identified using human antibody phage display that can recognize CD133 in both formalin-fixed tissues and cell lines. METHODS:A human single-chain variable fragment (scFv) antibody phage display library possessing a diversity of 8?×?109 was screened against fully glycosylated recombinant CD133. A counter screen was performed against deglycosylated CD133 to select for clones that preferentially recognized a glycosylation-independent epitope. The lead scFv was analyzed by flow cytometry and cloned into a rabbit immunoglobulin scaffold for immunohistochemistry (IHC). RESULTS:The antibody designated HA10 was found to bind a glycosylation-independent epitope on the peptide backbone of CD133 with high affinity. As a reagent for flow cytometry, HA10 detected CD133 more accurately than a commonly used commercially available antibody. IHC analysis with HA10 documented the staining of basal cells and luminal cells in healthy prostate sections. Weak staining of luminal cells was observed in adenocarcinoma sections at a very low frequency. Examination of a LuCaP patient-derived xenograft tissue microarray found that only three of the LuCaP models were positive for CD133. The three CD133pos LuCaP models all originated from non-AR driven metastatic prostate cancer with neuroendocrine differentiation. Subsequent interrogation of liver biopsies from a patient who failed second-generation anti-androgen therapy found high levels of CD133 staining. The original transurethral resection of the prostate from that patient was, however, absent of CD133. CONCLUSIONS:We have developed a novel antibody that was able to detect CD133 by both IHC and flow cytometry. Using HA10 as an IHC reagent, we found that CD133 is a marker for a very rare cell type in both healthy prostate and adenocarcinoma sections. Our preliminary investigation also suggests that there may be an association between CD133 and non-AR driven prostate cancer with neuroendocrine differentiation.
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Project description:Tumor necrosis factor alpha (TNF-?) is an inflammatory cytokine which plays crucial roles in pathogenesis of inflammatory diseases. The current study aimed to investigate the binding abilities of I44 and I49 domain antibodies to TNF-?. The dAbs were expressed in bacterial expression system and purified by affinity chromatography using Ni-sepharose column. The expression and purity of the proteins were evaluated using western blotting and SDS-PAGE techniques, respectively. ELISA experiment showed that I44 and I49 dAbs bind to TNF-? with the binding constants (Kd) of 5.18 ± 1.41 and 2.42 ± 0.55 µM, respectively. The inhibitory effect of dAbs on TNF-? biological effect was determined in MTT assay in which I44 and I49 prevented TNF-? cell cytotoxicity with IC50 values of 6.61 and 3.64 µM, respectively. The identified anti-TNF-? dAbs could bind to and inhibit TNF-? activity. The dAbs activities can be attributed to their ability to establish hydrogen bonds as well as hydrophobic contacts with TNF-?. The results of the current study can pave the way for further structural studies in order to introduce new more potent anti-TNF-? antibodies.
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