RESUMO
AQP4-IgG is an autoantibody associated with neuromyelitis optica spectroscopic disorder (NMOSD), a central nervous system inflammatory disease that requires early diagnosis and treatment. We designed two fusion proteins, AQP4-DARPin1 and AQP4-DARPin2, comprising the complete antigenic epitopes of aquaporin-4 (AQP4) and the constant region of the scaffold protein DARPin. These fusion proteins were expressed and purified from Escherichia coli and coated on microplates to develop an efficient method for detecting AQP4-IgG. Molecular dynamics simulation revealed that the fusion of AQP4 extracellular epitopes with DARPin did not alter the main structure of DARPin. The purified AQP4-DARPins bound recombinant antibody rAb-53 (AQP4-IgG) with affinities of 135 and 285 nM, respectively. Enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation demonstrated that AQP4-DARPin1 specifically recognized AQP4-IgG in the NMOSD patient serum. AQP4-DARPin1 as a coated antigen showed higher ELISA signal and end point dilution ratio than full-length AQP4. Our AQP4-DARPin1-coated AQP4-IgG ELISA had 100% specificity and 90% sensitivity. These results indicate that AQP4-DARPin1, compared to existing detection strategies that use full-length or extracellular loop peptides of AQP4, provides a new and more effective approach to the ELISA detection of NMOSD.
Assuntos
Neuromielite Óptica , Humanos , Neuromielite Óptica/diagnóstico , Proteínas de Repetição de Anquirina Projetadas , Aquaporina 4/genética , Epitopos , Imunoglobulina GRESUMO
Anti-aquaporin-4 autoantibodies (AQP4-IgG) are implicated in the pathogenesis of neuromyelitis optica spectrum disorders (NMOSD), and their removal from the blood circulation is considered to be an effective method for acute treatment. An ideal extracorporeal AQP4-IgG removal system should have high specificity, which means that it can selectively remove AQP4-IgG without affecting normal immunoglobulins. However, the conventional tryptophan immobilized column lacks sufficient specificity and cannot achieve this goal. In this study, we successfully prepared a fusion protein chimeric AQP4, which consists of the complete antigenic epitopes of human AQP4 and the constant region of scaffold protein DARPin. Chimeric AQP4 was expressed and purified from Escherichia coli, and then immobilized on agarose gel as a ligand for selective capture of AQP4-IgG immunosorbent. The prepared immunosorbent had a theoretical maximum adsorption capacity of 20.48 mg/g gel estimated by Langmuir isotherm. In vitro plasma perfusion tests demonstrated that the chimeric AQP4 coupled adsorbent had remarkable adsorption performance, and could eliminate more than 85 % of AQP4-IgG under the gel-to-plasma ratio of 1:50. Moreover, it exhibited high specificity because other human plasma proteins were not adsorbed in the dynamic adsorption experiment. These results suggest that the chimeric AQP4 coupled immunosorbent can provide a new approach for specific immunoadsorption (IA) treatment of NMOSD.
Assuntos
Aquaporina 4 , Neuromielite Óptica , Humanos , Aquaporina 4/genética , Imunoadsorventes , Neuromielite Óptica/terapia , Imunoglobulina G , EpitoposRESUMO
Removing excessively produced cytokines is of paramount significance in blood purification therapy for hypercytokinemia-associated diseases. In this study, we devised a conduit that is modified with nanobodies (Nb) and incorporates static mixers (Nb-SMC) to eliminate surplus cytokines from the bloodstream. The low-pressure-drop (LPD) static mixer, with each unit featuring two 90°-crossed blades, was strategically arranged in a tessellated pattern on the inner wall of the conduit to induce turbulent mixing effects during the flow of blood. This arrangement enhances mass transfer and molecular diffusion, thereby assisting in the identification and elimination of cytokines. By utilizing computational fluid dynamics (CFD) studies, the Nb-SMC was rationally designed and prepared, ensuring an optimal interval between two mixer units (H/G = 2.5). The resulting Nb-SMC exhibited a remarkable selective clearance of IL-17A, reaching up to 85 %. Additionally, the process of Nb immobilization could be adjusted to achieve the simultaneous removal of multiple cytokines from the bloodstream. Notably, our Nb-SMC displayed good blood compatibility without potential adverse effects on the composition of human blood. As the sole documented static mixer-integrated conduit capable of selectively eliminating cytokines at their physiological concentrations, it holds promise in the clinical potential for hypercytokinemia in high-risk patients. STATEMENT OF SIGNIFICANCE: High-efficient cytokines removal in critical care still remains a challenge. The conduit technique we proposed here is a brand-new strategy for cytokines removal in blood purification therapy. On the one hand, nanobody endows the conduit with specific recognition of cytokine, on the other hand, the build-in static mixer enhances the diffusion of antigenic cytokine to the ligand. The combination of these two has jointly achieved the efficient and specific removal of cytokine. This innovative material is the only reported artificial biomaterial capable of selectively eliminating multiple cytokines under conditions close to clinical practice. It has the potential to improve outcomes for patients with hypercytokinemia and reduce the risk of adverse events associated with current treatment modalities.
Assuntos
Citocinas , Hemoperfusão , Humanos , Hemoperfusão/métodos , Síndrome da Liberação de Citocina , Próteses e ImplantesRESUMO
Medical plastics such as those found in endotracheal tubes are widely used in intensive care units for the treatment of critically ill patients. Although commonplace in hospital environment, these catheters are at a high risk of bacterial contamination and have been found responsible for numerous health-care-associated infections. Antimicrobial coatings that can prevent harmful bacterial growth are required to reduce the occurrence of such infections. In this study, we introduce a facile surface treatment strategy that could form antimicrobial coatings on the surface of average medical plastics. The strategy involves treatment of activated surfaces with lysozyme, a natural antimicrobial enzyme presenting in human lacrimal gland secretions which is widely used for wound healing. Using ultra-high molecular weight polyethylene (UHMWPE) as the representative surface, oxygen/argon plasma treatment for 3 min led to the increase of surface roughness and the generation of negatively charged groups, with the zeta potential measured as -94.5 mV at pH 7. The activated surface could accommodate lysozyme with a density of up to 0.3 nmol/cm2 through electrostatic interaction. Antimicrobial activity of the resulting surface (UHMWPE@Lyz) was characterized with Escherichia coli and Pseudomonas sp. strains, and the treated surface significantly inhibited the bacterial colonization and the formation of biofilm compared to the untreated UHMWPE. This method of constructing an effective lysozyme-based antimicrobial coating is a generally applicable, simple and fast process for surface treatment with no adverse solvent and wastes involved.
Assuntos
Anti-Infecciosos , Muramidase , Humanos , Muramidase/farmacologia , Polietilenos/farmacologia , Polietilenos/química , Biofilmes , Anti-Infecciosos/farmacologia , BactériasRESUMO
Developing a facile, efficient, and versatile polyphenol coating strategy and exploring its novel applications are of great significance in the fields of material surfaces and interfaces. Herein, a one-step assembly strategy for constructing novel tannic acid (TA) coatings via a solvent evaporation method is reported using TA and polycyclodextrin (PCD) particles (TPP). TPP with a high phenolic group activity of 88% integrates the advantages of host-guest and polyphenol chemistry. The former can drive TPP dynamically assemble into a large and collective aggregation activated by high temperature or density, and the latter provides excellent adhesion properties to substrates (0.9 mg cm-2 ). TPP can assemble into a coating (TPC) rapidly on various substrates within 1 h at 37 °C while with a high availability of feed TPP (≈90%). The resulting TPC is not only high-temperature steam-sensitive for use as an anti-fake mask but also pH-sensitive for transforming into a free-standing film under physiological conditions. Moreover, various metal ions and functional particles can incorporate into TPC to extend its versatile properties including antibacterial activity, enhanced stability, and conductivity. This work expands the polyphenol coating strategy and builds up a one-step and efficient preparation platform of polyphenol coating for multiapplication prospects in various fields.
RESUMO
2,3-butanediol dehydrogenase (BDH, EC 1.1.1.76) also known as acetoin reductase (AR, EC 1.1.1.4) is the key enzyme converting acetoin (AC) into 2,3-butanediol (BD) and undertaking the irreversible conversion of diacetyl to acetoin in various microorganisms. The existence of three BDHs (R,R-, meso-, and S,S-BDH) product different BD isomers. Catalyzing mechanisms of meso- and S,S-BDH have been understood with the assistance of their X-ray crystal structures. However, the lack of structural data for R,R-BDH restricts the integral understanding of the catalytic mechanism of BDHs. In this study, we successfully crystallized and solved the X-ray crystal structure of Bacillus subtilis R,R-BDH. A zinc ion was found locating in the catalytic center and coordinated by Cys37, His70 and Glu152, helping to stabilize the chiral substrates observed in the predicted molecular docking model. The interaction patterns of different chiral substrates in the molecular docking model explained the react priority measured by the enzyme activity assay of R,R-BDH. Site-directed mutation experiments determined that the amino acids Cys37, Thr244, Ile268 and Lys340 are important in the catalytically active center. The structural information of R,R-BDH presented in this study accomplished the understanding of BDHs catalytic mechanism and more importantly provides useful guidance for the directional engineering of R,R-BDH to obtain high-purity monochiral BD and AC.
Assuntos
Acetoína , Bacillus subtilis , Bacillus subtilis/metabolismo , Acetoína/metabolismo , Simulação de Acoplamento Molecular , Oxirredutases do Álcool/metabolismoRESUMO
Environment-sensitive fluorogenic antibodies enable target-specific bioimaging with reduced unspecific background signal and improved spatiotemporal resolution. However, current strategies for the construction of fluorogenic antibodies are hard to handle due to challenges that lie in the prior design of fluorogenic probes and subsequent antibody labeling. Here, we report a simple strategy to generate a fluorogenic nanobody, which we term D-body, by in situ incorporation of a reduction-responsive Nile blue foldamer which is self-quenched via a dimerization-caused quenching mechanism. The D-body can be efficiently internalized by cells with high epidermal growth factor receptor expression levels and is highly fluorogenic upon lysosomal activation, allowing wash-free cell imaging with exquisite specificity and fast in vivo imaging with a high tumor-to-background ratio. The modular D-body is readily available and easy to handle, offering a platform that is highly tunable for bioimaging applications.
Assuntos
Corantes Fluorescentes , Neoplasias , Anticorpos de Domínio Único , Humanos , Neoplasias/diagnóstico por imagemRESUMO
Bispecific antibodies (bis-Nbs) have been extensively developed since the concept was devised over the decades. Taking advantage of the superior characteristics of nanobodies, bis-Nbs exhibit an emerging tendency to become the new generation of research and diagnostic tools. Traditional strategies to connect the homo- or heterogeneous monomers are commonly applied, but there are still technical issues to generate the bispecific molecules as efficiently as designed. Here, we utilize SnoopLigase to directly tether the C terminus (C-C) of the tagged nanobodies against tumor necrosis factor-α (TNF-α) and interleukin-17A (IL-17A). Under optimal conditions, the yield of C-C-linked bis-Nbs can reach as high as 70% due to the existence of SnoopLigase. The prepared bis-Nbs possessed similar or even higher affinity as the monomers and significantly inhibited the proliferation and migration of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) induced by TNF-α and IL-17A. This study provides an innovative route for using SnoopLigase to realize a highly efficient generation of C-C-linked bis-Nbs. The approach can be applied to different and multicomponent systems for their potential applications in disease diagnosis and treatment.
Assuntos
Anticorpos Biespecíficos , Artrite Reumatoide , Anticorpos de Domínio Único , Sinoviócitos , Anticorpos Biespecíficos/farmacologia , Artrite Reumatoide/tratamento farmacológico , Células Cultivadas , Fibroblastos , Humanos , Interleucina-17 , Anticorpos de Domínio Único/farmacologia , Membrana Sinovial/patologia , Sinoviócitos/patologia , Fator de Necrose Tumoral alfaRESUMO
Camelid-derived nanobody is emerging as a resourceful platform for developing immunoaffinity ligands for chromatography applications. Featured by high affinity and selectivity, BC2 nanobody (BC2-Nb), which can recognize a specific epitope tag (PDRKAAVSHWQQ, termed BC2T), is potential to be developed as a general tool for recombinant protein purification. However, excessively high affinity between binding partners makes the desorption of products less efficient and limits its application. Aiming to improve elution efficiency, structure-guided mutations of BC2-Nb were conducted to adjust the structural flexibility of its antigen-binding site. Six ligand variants were obtained with their binding affinity decreasing by about 100-fold. Among them, one mutated BC2-Nb named 44D was chosen to prepare immunoaffinity resin, and its adsorption and elution performance were well characterized. The site-directed mutation led to the equilibrium dissociation constant (KD) of BC2-Nb changing from 1.4 × 10-9 M to 1.4 × 10-7 M (44D). The resin using 44D as ligand retained a static binding capacity of 19.14 mg/mL toward BC2T-fused enhanced green fluorescent protein (eGFP-BC2T). Significantly improved elution efficiency was obtained with the mutated ligand. Protein recovery reached 94% at pH 3.5 for 44D-based resin, while the resin based on original BC2-Nb could only achieve its highest recovery of 84% at pH 2. In addition, a neutral elution condition (1 M arginine containing 50% propylene glycol, pH 7.4) was also found effective, which allowed a product recovery of 95%. The resin enabled direct capturing of eGFP-BC2T from bacterial lysates, and the one-step purification with the both elution conditions could achieve a product purity of more than 90%. This study provided a promising affinity ligand, and also proved the feasibility of controlling the elution process of nanobody-based affinity resin through the strategy of binding sites modification.
Assuntos
Peptídeos , Adsorção , Cromatografia de Afinidade/métodos , Ligantes , Peptídeos/química , Proteínas RecombinantesRESUMO
We here found that while Meldrum's acid as the reactive warhead allows for the attachment of a single chemical modification on aldehyde-containing proteins, pyrazolone derivatives in combination with a phosphine nucleophile enable protein dual site-specific conjugation with the same or distinct moieties. These reactions are efficient and convergent under biocompatible conditions and allow access to protein bioconjugates with superior stability, homogeneity and flexibility. Our work expands the repertoire of bioconjugation chemistries and offers opportunities to construct bioconjugates with defined structure that have potential for medical and biomaterial applications.
Assuntos
Carbono , Proteínas , Aldeídos , Materiais Biocompatíveis/química , Carbono/química , Proteínas/químicaRESUMO
Tyrosine, a simple and well-available natural amino acid, is featured by the small size of the compound that contains multiple reactive groups. This study developed an efficient bioconjugation strategy using tyrosine-based dual-functional interfaces. When tyrosine molecules are immobilized on the surface of a supporting material through amino groups, their carboxyl groups can function as an attracting trap due to their anionic nature at neutral pH and ability to chelate nickel(II) ions (Ni2+), allowing the capture and enrichment of cationic proteins and histidine (His)-tagged proteins on the surface. The trapped proteins can be further covalently immobilized on site through ruthenium-mediated photochemical cross-linking, which has been found to be highly efficient and can be completed within minutes. This strategy was successfully applied to two different material systems. We found that tyrosine-modified agarose beads had a binding capacity of the His-tagged enhanced green fluorescent protein comparable to that of commonly used nitrilotriacetic acid-based resins, and further covalent coupling via dityrosine cross-linking achieved a yield of 85% within 5 min, without compromising much on its fluorescence activity. On the surface of tyrosine-modified 316L stainless steel, lysozyme was captured through electrostatic interaction and further immobilized. The resultant surface exhibited remarkable antibacterial activity against both Staphylococcus aureus and Escherichia coli. Such a tyrosine-based capture-then-coupling method is featured by its simplicity, high coupling efficiency, and high utilization rate of target molecules, making it particularly suitable for the proteins that are highly priced or vulnerable to general immobilization chemistry.
Assuntos
Histidina , Ácido Nitrilotriacético , Histidina/química , Indicadores e Reagentes , Níquel/química , Ácido Nitrilotriacético/química , Tirosina/químicaRESUMO
The presence of unique heavy chain-only antibodies (HCAbs) in camelids was discovered at Vrije Universiteit Brussel (VUB, Brussels, Belgium) at a time when many researchers were exploring the cloning and expression of smaller antigen-binding fragments (Fv and Fab) from hybridoma-derived antibodies. The potential importance of this discovery was anticipated, and efforts were immediately undertaken to understand the emergence and ontogeny of these HCAbs as well as to investigate the applications of the single-domain antigen-binding variable domains of HCAbs (nanobodies). Nanobodies were demonstrated to possess multiple biochemical and biophysical advantages over other antigen-binding antibody fragments and alternative scaffolds. Today, nanobodies have a significant and growing impact on research, biotechnology, and medicine.
Assuntos
Camelídeos Americanos , Anticorpos de Domínio Único , Animais , Anticorpos/química , Biotecnologia , Cadeias Pesadas de Imunoglobulinas , Anticorpos de Domínio Único/químicaRESUMO
Nanobodies (Nbs) can be successfully retrieved following phage, bacterial, yeast, or ribosome display of immune, synthetic, or naïve libraries. However, after panning, multiple individual Nb clones need to be screened and assessed for solubility, antigen specificity, affinity, and potential biological function. Therefore, it is highly desirable to have a convenient expression strategy to obtain sufficient protein for in-depth characterization of the Nbs. The presence of a purification and detection tag, as well as a chemically reactive group to enable simple generation of Nb derivatives, would be of great help in this regard. Here, we provide a general protocol for high yield cytoplasmic expression and purification of formylglycine generating enzyme (FGE)-tagged Nbs. The cysteine within the FGE tag is easily converted to formylglycine by passing the FGE-tag containing Nb over a continuous-flow bio-catalysis system. The aldehyde group within the formylglycine side chain at the C-terminal end of the Nb is suitably located for subsequent bio-orthogonal reactions to fluorescent dyes, biotin, polyethylene glycol, or chromatography resins. We also include methods for production of high yield recombinant FGE, as well as conditions for its immobilization on Sepharose to produce the continuous-flow bio-catalysis system.
Assuntos
Aldeídos , Anticorpos de Domínio Único , Aldeídos/química , Cisteína/química , Glicina/análogos & derivados , Glicina/metabolismoRESUMO
Here, we report a new protocol for enhancing the soluble expression of inclusion body (IB)-prone proteins in E. coli using nanobodies (Nbs) as a molecular-specific chaperone. The specific intracellular binding between the cognate-Nbs and the antigen is successfully achieved and enables the formation of a soluble Nb-antigen complex in E. coli. We further expand this method by adding an epitope tag (EPEA-tag) to the target proteins, and the anti-EPEA Nb was intended to act as the chaperone for in vivo binding with the EPEA tag. Such substitution may develop a "multi-specific" Nb-chaperone that can simultaneously and effectively cope with different IB proteins of interest.
Assuntos
Proteínas de Escherichia coli/química , Corpos de Inclusão/química , Chaperonas Moleculares/química , Anticorpos de Domínio Único/química , SolubilidadeRESUMO
The rapid development of bioeconomy urgently needs the support of biotechnology talents. Establishing an innovative training mode of biotechnology talents can provide support for regional economic development and industrial upgrading. Closely revolved around the concepts of new engineering disciplines development, such as serving the national strategy, docking industry, leading the future development and student-centered, a new economy-oriented training system was developed in School of Bioengineering of Dalian University of Technology. These systems include interdisciplinary curriculum system reconstruction, project-based teaching mode reform, evaluation system implementation and other aspects. The reform and exploration of the first-class biotechnology major under the new economic situation, puts forward the theory of value guidance, deep foundation, strong sense of innovation, technical and non-technical core ability literacy. This reform meets the industry demand for talent diversification, personalization, and dynamic change, helps the merge of industry and education, which provides a way for fostering first-class biotechnology-majored undergraduates.
Assuntos
Bioengenharia , Biotecnologia , Humanos , Engenharia Biomédica , Estudantes , CurrículoRESUMO
Elimination of overproduced cytokines from blood can relieve immune system disorders caused by hypercytokinemia. Due to the central roles of interleukin-17A (IL-17A) plays in regulating the immunity and inflammatory responses in humans, here, a novel immunosorbent containing anti-IL-17A nanobodies (Nbs) was constructed for IL-17A removal from blood. The theoretical maximum adsorption capacity estimated from the Langmuir isotherm is up to 11.55 mg/g gel, which is almost consistent with the saturated adsorption capacity determined in dynamic adsorption. The in vitro plasma perfusion test demonstrated a remarkable adsorptive performance of the Nb-coupled sorbent since more than 75% IL-17A could be eliminated under the plasma/sorbent ratio of 1000:1. These results indicated the Nb-loaded immunosorbent can provide a simple and economic platform technology for immunoaffinity depletion of single or even multiple cytokines from plasma.
Assuntos
Análise Química do Sangue , Imunoadsorventes , Interleucina-17 , Análise Química do Sangue/métodos , Humanos , Imunoadsorventes/química , Interleucina-17/sangue , Interleucina-17/isolamento & purificação , Anticorpos de Domínio Único/metabolismoRESUMO
Mixed-mode chromatography provides a promising strategy for industrial protein purification for its potential merit of balancing efficiency and cost-effectiveness. However, mixed-mode media with satisfactory selectivity and binding capacity towards antibody are still urgently needed. A new type of mixed-mode chromatography resin was prepared using benzotriazole-5-carboxylicas as ligand (BTA MM), and its application in antibody separation was explored. A typical pH-dependent protein binding was observed, and the neutral condition was favorable for antibody adsorption. Dynamic binding capacity of human immunoglobulin G (hIgG) was 57.7 mg/mL at pH 7.4 (10 mM phosphate buffer, containing 150 mM NaCl), while elution with acidic solutions (pH 3-4) could achieve a recovery of more than 85%. Protein adsorption on the resin showed a salt-independent manner, thus it could work under physiological solution conditions, with satisfied antibody selectivity. One-step purification of antibody components from human serum samples could obtain a product with the purity more than 84%. Satisfied performance was also observed when the adsorbent was used for purifying a IgG1-type monoclonal antibody (mAb) from cell culture supernatant. In addition, the benzotriazole adsorbent has been found stable enough to withstand autoclave sterilization and other harsh conditions, including 1 M NaOH, 1 M HCl, and 75% ethanol. The results proved the potential of this type of mixed-mode chromatography medium for industrial antibody purification.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Cromatografia Líquida/métodos , Triazóis/química , Adsorção , Animais , Anticorpos Monoclonais/química , Células CHO , Cricetinae , Cricetulus , Humanos , Imunoglobulina G/isolamento & purificação , LigantesRESUMO
VHH-based immunosorbents are an emerging and promising tool for the removal of toxic substances from plasma. However, the small size of VHHs is a double-edged sword, bringing both benefits and drawbacks to the immunosorbent. The small size of the VHH allows a higher coupling density, while the closer distance to the resin might create steric hindrance for paratope access. The latter could be avoided by inserting a linker between the VHH and the gel attachment site. Here, we report an approach to improve the activity retention of the immobilized VHH by selecting suitable linkers between the VHH and the site-specific immobilization site on the resin. Seven peptide linkers differing in length and flexibility were fused to the VHH and contained the formylglycine generating enzyme (FGE) recognition sequence. These constructs were expressed in the cytoplasm of bacteria and purified, the VHH production yield and affinity for its cognate antigen was measured. Furthermore, the fGly conversion, the immobilization of the aldehyde-containing nanobodies, the immobilization on resin and the antigen binding activity of the VHH-based immunoadsorbents was monitored. The VHH with longer and rigid, proline-rich linkers exhibited good expression yield of approximately 160 mg/L of culture, a fGly conversion of up to 100%, and the highest activity retention rate of more than 68%. This study unveiled two suitable linkers for the preparation of VHH-based immunosorbents that will assist the development of their clinical application.
Assuntos
Imunoadsorventes/química , Peptídeos/química , Anticorpos de Domínio Único/químicaRESUMO
Affinity chromatography is generally regarded as a powerful tool allowing the single step purification of recombinant proteins with high purity and yields. However, for most protein products, affinity purification methods for industrial applications are not readily available, mainly due to the lack of specific and robust natural counterparts that could function as affinity ligands. In this study, we explored the applicability of nanobody-based peptide-tag immunorecognition systems as a platform for affinity chromatography. Two typical nanobodies (BC2-nb and Syn2-nb) that are capable of recognizing specifically a particular peptide-tag, were prepared through prokaryotic expression and proved to be able to bind with nanomolar affinity to their cognate tag fused to enhanced green fluorescent protein (eGFP). Through an epoxy-based immobilization reaction, the two nanobodies were coupled on a Sepharose CL-6B matrix under the same conditions. The remaining antigen binding activity of the immobilized BC2-nb and Syn2-nb was determined to be 83.1% and 42.9%, yielding the resins with the dynamic binding capacity (DBC) of 21.4 mg/mL and 5.9 mg/mL, respectively. The immobilized affinity ligands exhibited high binding specificity towards their respective target peptides, yielding a product purity above 90% directly from crude bacterial lysates in one single chromatographic step. However, for the both affinity complexes, desorption has been found difficult, and effective recovery of the bound products could be only achieved with competitive elution or after employing harsh conditions such as 10 mM NaOH solution, which will compromise the reuse cycles of the affinity resins. This study shows the potential of nanobody-based affinity chromatography for efficient purification of recombinant proteins especially from complex feedstocks and reveals the primary issues to be addressed to develop a successful application.
Assuntos
Cromatografia de Afinidade/métodos , Peptídeos/imunologia , Anticorpos de Domínio Único/imunologia , Sequência de Aminoácidos , Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Cromatografia Líquida de Alta Pressão , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cinética , Peptídeos/química , Peptídeos/isolamento & purificação , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/metabolismoRESUMO
Removing ß2-microglobulin (ß2M) from blood circulation is considered to be the most effective method to delay the occurrence of dialysis-related amyloidosis (DRA). The ideal extracorporeal ß2M removal system should be cost-effective, highly specific and having a high capacity. However, the traditional technologies based on size exclusion do not have an adequate specificity, and alternative immunosorbents have limited applications due to low capacity and their high cost. Nanobodies (Nbs), the smallest functional recombinant antibody fragments, offer several advantages to overcome these obstacles. In this study, an anti-ß2M Nb with a C-terminal thiol-tag was successfully prepared from E. coli for site-directed and oriented immobilization and usage as capture ligand in a ß2M-selective immunosorbent. The prepared immunosorbent showed a high binding capacity of up to 7 mg ß2M per mL resin, which is 17 times higher than that of previous studies using single-chain variable antibody fragments (scFv). Furthermore, an exceptional high specificity has been demonstrated as other human serum proteins were not adsorbed during dynamic adsorption experiments. About 80% of the original binding capacity of the immunosorbent was restored after four consecutive easy regenerations, whereas 90% of the original capacity was retained after 1-month storage of the resin. Moreover, the mathematical model fitted very well the in vitro perfusion. The results with this pioneering immunosorbent confirm its possible clinical application and is expected to reach the required clinical effect of immunoadsorption therapy. STATEMENT OF SIGNIFICANCE: Dialysis-related amyloidosis (DRA), associated with the accumulation of ß2-microglobulin (ß2M), is a serious complication of end-stage kidney disease. Removing ß2M from blood circulation by extracorporeal blood purification is considered to be the most effective method to delay the occurrence of DRA. However, the existing methods are incapable to eliminate sufficient quantities of ß2M from circulation, either because of lack of specificity, high cost or for low capacity. In this manuscript, we provide a practical and economic immunosorbent based on anti-ß2M nanobody for DRA. The prepared immunosorbent was reusable and storable, and demonstrated high specificity and realized a high binding capacity of up to 7 mg ß2M per mL resin, which is 17 times higher than that of the previous studies.