RESUMEN
A novel tandem affinity tag is presented that enables the use of cation exchange resins for initial affinity purification, followed by an additional column step for enhanced purity and affinity tag self-removal. In this method, the highly charged heparin-binding tag binds strongly and selectively to either a strong or weak cation exchange resin based on electrostatic interactions, effectively acting as an initial affinity tag. Combining the heparin-binding tag (HB-tag) with the self-removing iCapTag™ provides a means for removing both tags in a subsequent self-cleaving step. The result is a convenient platform for the purification of diverse tagless proteins with a range of isoelectric points and molecular weights. In this work, we demonstrate a dual column process in which the tagged protein of interest is first captured from an E. coli cell lysate using a cation exchange column via a fused heparin-binding affinity tag. The partially purified protein is then diluted and loaded onto an iCapTag™ split-intein column, washed, and then incubated overnight to release the tagless target protein from the bound tag. Case studies are provided for enhanced green fluorescent protein (eGFP), beta galactosidase (ßgal), maltose binding protein (MBP) and beta lactamase (ßlac), where overall purity and host cell DNA clearance is provided. Overall, the proposed dual column process is shown to be a scalable platform technology capable of accessing both the high dynamic binding capacity of ion exchange resins and the high selectivity of affinity tags for the purification of recombinant proteins.
Asunto(s)
Escherichia coli , Heparina , Proteínas Recombinantes de Fusión/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/química , Cromatografía de Afinidad/métodos , Heparina/metabolismoRESUMEN
Current biological research requires simple protein bioseparation methods capable of purifying target proteins in a single step with high yields and purities. Conventional affinity tag-based approaches require specific affinity resins and expensive proteolytic enzymes for tag removal. Purification strategies based on self-cleaving aggregating tags have been previously developed to address these problems. However, these methods often utilize C-terminal cleaving contiguous inteins which suffer from premature cleavage, resulting in significant product loss during protein expression. In this work, we evaluate two novel mutants of the Mtu RecA ΔI-CM mini-intein obtained through yeast surface display for improved protein purification. When used with the elastin-like-polypeptide (ELP) precipitation tag, the novel mutants - ΔI-12 and ΔI-29 resulted in significantly higher precursor content, product purity and process yield compared to the original Mtu RecA ΔI-CM mini-intein. Product purities ranging from 68 % to 94 % were obtained in a single step for three model proteins - green fluorescent protein (GFP), maltose binding protein (MBP) and beta-galactosidase (beta-gal). Further, high cleaving efficiency was achieved after 5 h under most conditions. Overall, we have developed improved self-cleaving precipitation tags which can be used for purifying a wide range of proteins cheaply at laboratory scale.
Asunto(s)
Inteínas , Proteínas de Unión a Maltosa , Rec A Recombinasas , beta-Galactosidasa , Inteínas/genética , beta-Galactosidasa/genética , beta-Galactosidasa/química , beta-Galactosidasa/aislamiento & purificación , beta-Galactosidasa/metabolismo , Proteínas de Unión a Maltosa/genética , Proteínas de Unión a Maltosa/química , Proteínas de Unión a Maltosa/metabolismo , Rec A Recombinasas/genética , Rec A Recombinasas/química , Rec A Recombinasas/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Elastina/química , Elastina/genética , Elastina/aislamiento & purificación , Precipitación Química , Escherichia coli/genética , Escherichia coli/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/químicaRESUMEN
A purification system was constructed with the N-segment of the Npu DnaE split intein as an affinity ligand immobilized onto an epoxy-activated medium and the C-segment used as the cleavable tag fusing target protein. The affinity properties of C-tagged proteins adsorbed on IN affinity chromatography medium were studied with GFP as a model target protein. The saturated adsorption capacity and dynamic adsorption capacity reached 51.9-21.0 mg mL-1, respectively. With this system, two model proteins, GFP and alcohol dehydrogenase (ADH), has been successfully taglessly purified with regulation of Zn2+ and DTT. The yield, purification factor and purity of purified tagless GFP reached 39, 11.7 and 97%, respectively; while these values for purified tagless ADH were 38.2, 6.8 and 91%, respectively. These results showed that the system for Npu DnaE split intein-mediated affinity adsorption and in situ cleavage is a potential platform for recombinant protein production.
Asunto(s)
Biotecnología/métodos , Cromatografía de Afinidad/instrumentación , Cromatografía de Afinidad/métodos , Proteínas Recombinantes/aislamiento & purificación , Alcohol Deshidrogenasa/química , ADN Polimerasa III/genética , ADN Polimerasa III/metabolismo , Ditiotreitol/química , Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes/química , Inteínas , Ligandos , Dominios Proteicos , Empalme de Proteína , Proteínas Recombinantes/química , Zinc/químicaRESUMEN
Many diseases and disorders are linked to exposure to endocrine disrupting chemicals (EDCs) that mimic the function of natural estrogen hormones. Here we present a Rapid Adaptable Portable In-vitro Detection biosensor platform (RAPID) for detecting chemicals that interact with the human estrogen receptor ß (hERß). This biosensor consists of an allosteric fusion protein, which is expressed using cell-free protein synthesis technology and is directly assayed by a colorimetric response. The resultant biosensor successfully detected known EDCs of hERß (BPA, E2, and DPN) at similar or better detection range than an analogous cell-based biosensor, but in a fraction of time. We also engineered cell-free protein synthesis reactions with RNAse inhibitors to increase production yields in the presence of human blood and urine. The RAPID biosensor successfully detects EDCs in these human samples in the presence of RNAse inhibitors. Engineered cell-free protein synthesis facilitates the use of protein biosensors in complex sample matrices without cumbersome protein purification.
Asunto(s)
Técnicas Biosensibles/métodos , Sistema Libre de Células/metabolismo , Disruptores Endocrinos/sangre , Disruptores Endocrinos/orina , Biosíntesis de Proteínas/fisiología , Sistema Libre de Células/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Disruptores Endocrinos/farmacología , Receptor beta de Estrógeno/antagonistas & inhibidores , Receptor beta de Estrógeno/metabolismo , Humanos , Biosíntesis de Proteínas/efectos de los fármacosRESUMEN
The use of cell-free systems to produce recombinant proteins has grown rapidly over the past decade. In particular, cell-free protein synthesis (CFPS) systems based on mammalian cells provide alternative methods for the production of many proteins, including those that contain disulfide bonds, glycosylation, and complex structures such as monoclonal antibodies. In the present study, we show robust production of turbo green fluorescent protein (tGFP) and streptokinase in a cell-free system using instrumented mini-bioreactors for highly reproducible protein production. We achieved recombinant protein production (â¼600 µg/ml of tGFP and 500 µg/ml streptokinase) in 2.5 hr of expression time, comparable to previously reported yields for cell-free protein expression. Also, we demonstrate the use of two different affinity tags for product capture and compare those to a tag-free self-cleaving intein capture technology. The intein purification method provided a product recovery of 86%, compared with 52% for conventionally tagged proteins, while resulting in a 30% increase in total units of activity of purified recombinant streptokinase compared with conventionally tagged proteins. These promising beneficial features combined with the intein technology makes feasible the development of dose-level production of therapeutic proteins at the point-of-care.
Asunto(s)
Sistema Libre de Células , Mezclas Complejas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Estreptoquinasa/biosíntesis , Estreptoquinasa/aislamiento & purificación , Animales , Células CHO , Cricetulus , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/aislamiento & purificación , Proteínas Recombinantes/genética , Estreptoquinasa/genéticaRESUMEN
Cell-Free Protein Synthesis (CFPS) offers many advantages for the production of recombinant therapeutic proteins using the CHO cell-free system. However, many complex proteins are still difficult to express using this method. To investigate the current bottlenecks in cell-free glycoprotein production, we chose erythropoietin (40% glycosylated), an essential endogenous hormone which stimulates the development of red blood cells. Here, we report the production of recombinant erythropoietin (EPO) using CHO cell-free system. Using this method, EPO was expressed and purified with a twofold increase in yield when the cell-free reaction was supplemented with CHO microsomes. The protein was purified to near homogeneity using an ion-metal affinity column. We were able to analyze the expressed and purified products (glycosylated cell-free EPO runs at 25-28 kDa, and unglycosylated protein runs at 20 kDa on an SDS-PAGE), identifying the presence of glycan moieties by PNGase shift assay. The purified protein was predicted to have â¼2,300 IU in vitro activity. Additionally, we tested the presence and absence of sugars on the cell-free EPO using a lectin-based assay system. The results obtained in this study indicate that microsomes augmented in vitro production of the glycoprotein is useful for the rapid production of single doses of a therapeutic glycoprotein drug and to rapidly screen glycoprotein constructs in the development of these types of drugs. CFPS is useful for implementing a lectin-based method for rapid screening and detection of glycan moieties, which is a critical quality attribute in the industrial production of therapeutic glycoproteins.
Asunto(s)
Biotecnología/métodos , Sistema Libre de Células , Eritropoyetina/metabolismo , Microsomas/metabolismo , Proteínas Recombinantes/metabolismo , Animales , Células CHO , Cromatografía de Afinidad , Cricetulus , Electroforesis en Gel de Poliacrilamida , Eritropoyetina/química , Eritropoyetina/genética , Eritropoyetina/aislamiento & purificación , Expresión Génica , Glicosilación , Humanos , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Here we introduce a Rapid Adaptable Portable In vitro Detection biosensor platform (RAPID) for detecting ligands that interact with nuclear hormone receptors (NHRs). The RAPID platform can be adapted for field use, allowing rapid evaluation of endocrine disrupting chemicals (EDCs) presence or absence in environmental samples, and can also be applied for drug screening. The biosensor is based on an engineered, allosterically activated fusion protein, which contains the ligand binding domain from a target NHR (human thyroid receptor ß in this work). In vitro expression of this protein using cell-free protein synthesis (CFPS) technology in the presence of an EDC leads to activation of a reporter enzyme, reported through a straightforward colorimetric assay output. In this work, we demonstrate the potential of this biosensor platform to be used in a portable "just-add-sample" format for near real-time detection. We also demonstrate the robust nature of the cell-free protein synthesis component in the presence of a variety of environmental and human samples, including sewage, blood, and urine. The presented RAPID biosensor platform is significantly faster and less labor intensive than commonly available technologies, making it a promising tool for detecting environmental EDC contamination and screening potential NHR-targeted pharmaceuticals.
Asunto(s)
Técnicas Biosensibles , Disruptores Endocrinos/análisis , Proteínas Recombinantes de Fusión/síntesis química , Receptores beta de Hormona Tiroidea/química , Evaluación Preclínica de Medicamentos , Humanos , Ligandos , Proteínas Recombinantes de Fusión/químicaRESUMEN
The discovery of inteins in the early 1990s opened the door to a wide variety of new technologies. Early engineered inteins from various sources allowed the development of self-cleaving affinity tags and new methods for joining protein segments through expressed protein ligation. Some applications were developed around native and engineered split inteins, which allow protein segments expressed separately to be spliced together in vitro. More recently, these early applications have been expanded and optimized through the discovery of highly efficient trans-splicing and trans-cleaving inteins. These new inteins have enabled a wide variety of applications in metabolic engineering, protein labeling, biomaterials construction, protein cyclization, and protein purification.
Asunto(s)
Inteínas/genética , Empalme de Proteína/genética , Proteínas/genética , Trans-Empalme/genética , Ingeniería de Proteínas/métodos , Ingeniería de Proteínas/tendencias , Proteínas/química , Proteínas/aislamiento & purificación , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Coloración y Etiquetado/métodos , Coloración y Etiquetado/tendenciasRESUMEN
Many important functional proteins often exhibit toxicity when overexpressed in heterologous hosts. Unfortunately, this toxicity can complicate the production of these proteins in recombinant systems, which can slow their characterization. Although a number of engineered expression strains and plasmids have been developed to optimize toxic protein expression, many targets remain recalcitrant in these systems due to extreme toxicity to the expression host. In this work, we have developed a novel protein purification platform based on intein trans-splicing, with special relevance for proteins that are extremely toxic to recombinant host cells. The toxic protein is split into two inactive fragments, which are separately expressed in fusion to the segments of a split intein. The N-terminal intein segment is first immobilized onto an affinity column and washed, followed by addition of the C-terminal segment and purification of the complex. The assembled intein controllably splices to deliver the mature target protein, simultaneously releasing the purified target from the affinity column. To optimize this method, we generated a hybrid split intein consisting of the N-terminus of the Npu DnaE intein and the C-terminus of the Ssp DnaE intein. This hybrid intein tolerates a wider range of amino acids at the +2 site of the C-terminal splicing junction than the Npu intein alone. In the production of the highly toxic homing endonuclease I-TevI, the yield from the hybrid intein is 50 % higher than the native Npu DnaE intein, while the I-TevI protein purified from both inteins showed native activity.
Asunto(s)
Inteínas/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/toxicidad , Trans-Empalme , Plásmidos , Proteínas Recombinantes/genéticaRESUMEN
Biologics and vaccines have been successfully developed over the last few decades to treat many diseases. Each of these drugs must be highly purified for clinical use. Monoclonal antibodies (mAbs), the dominant therapeutic modality on the market, can be easily purified using the standard Protein A affinity platform. However, no generally applicable affinity platforms are available for the manufacture of other therapeutic proteins for clinical use. Thus, multicolumn chromatography processes for widely being used for product purification. These processes demand significant optimization to meet desired product quality attributes, where each step also decreases final yields. In this work, we demonstrate the novel self-removing iCapTag™ affinity tag, which provides a new platform for capturing, concentrating, and purifying recombinant proteins. Importantly, this system provides a tagless target protein, which is suitable for research and clinical use, where the only requirement for tag removal is a small change in buffer pH. No additional proteins, reagents or cofactors are required. We also present case studies demonstrating the use of iCapTag™ for highly efficient purification of untagged interferon alpha 2b, the ML39 single chain variable fragment (scFv), and the receptor binding domain (RBD) of SARS-CoV-2 spike protein. These proteins were expressed and secreted by Expi293 cells with the self-removing tag fused to their N-terminus. We were able to obtain highly pure (> 99 %) tagless protein in a single purification step with high clearance of host cell DNA, tagged precursor, higher and lower molecular weight impurities. Based on these preliminary results, we propose the iCapTag™ as a universal capture platform for diverse classes of recombinant therapeutic proteins.
Asunto(s)
Cromatografía de Afinidad , Inteínas , Interferón-alfa , Proteínas Recombinantes , Humanos , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/química , Cromatografía de Afinidad/métodos , Interferón-alfa/aislamiento & purificación , Interferón-alfa/química , Interferón-alfa/genética , Anticuerpos de Cadena Única/aislamiento & purificación , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/genética , Animales , Interferón alfa-2/aislamiento & purificación , Glicoproteína de la Espiga del Coronavirus/aislamiento & purificación , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Cricetulus , Células CHO , SARS-CoV-2/genética , SARS-CoV-2/químicaRESUMEN
The rapid production of purified recombinant proteins has become increasingly important for countless applications. Many purification methods involve expression of target proteins in fusion to purification tags, which often must be removed from the target proteins after purification. Recently, engineered inteins have been used to create convenient self-cleaving tags for tag removal. Although intein methods can greatly simplify protein purification, commercially available expression vectors still rely on conventional restriction/ligation cloning methods for target gene insertion. We have streamlined this process by introducing Ligation-Independent Cloning (LIC) capability to our intein expression plasmids, which provides a simple method for constructing self-cleaving tag-target gene fusions. In this work, we demonstrate efficient gene insertion via this system, as well as target protein expression and purification consistent with previously reported results. Through this newly developed system, arbitrary protein genes can be rapidly incorporated into self-cleaving tag expression vectors, and their products purified using convenient platform methods.
Asunto(s)
Cromatografía de Afinidad/métodos , Clonación Molecular/métodos , Inteínas , Proteínas Recombinantes de Fusión/aislamiento & purificación , Secuencia de Bases , Quitina/química , Electroforesis en Gel de Poliacrilamida , Ensayos Analíticos de Alto Rendimiento , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Self-cleaving elastin-like protein (ELP) tags provide a very promising tool for recombinant protein purification. With this method, the target protein is purified by simple ELP-mediated precipitation steps, followed by self-cleavage and removal of the ELP tag. Unfortunately, however, inteins usually experience some level of pre-cleavage during protein expression, which can significantly decrease final yields. In this study, we solve this problem by splitting the intein into two ELP-tagged segments. Each segment is incapable of pre-cleavage alone, but the assembled segments release the target protein rapidly when assembled in vitro. The result is the very tight control of the tag cleaving reaction, combined with the simplicity of the ELP purification method. Using this system, we successfully purified four different sizes of target proteins with final yields comparable to or higher than our original contiguous intein-ELP system. Further, we demonstrate a streamlined split intein method, where cells expressing the tagged intein segments are combined prior to cell lysis, allowing the segments to be co-purified in a single reaction mixture.
Asunto(s)
Elastina/metabolismo , Inteínas/fisiología , Proteínas Recombinantes de Fusión/metabolismo , Biotecnología , Elastina/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Inteínas/genética , Proteínas Recombinantes de Fusión/genéticaRESUMEN
To study the effects and possible mechanisms of suspected endocrine disrupting compounds (EDCs), a wide variety of assays have been developed. In this work, we generated engineered Escherichia coli biosensor strains that incorporate the ligand-binding domains (LBDs) of the ß-subtype estrogen receptors (ERß) from Solea solea (sole), and Sus scrofa (pig). These strains indicate the presence of ligands for these receptors by changes in growth phenotype, and can differentiate agonist from antagonist and give a rough indication of binding affinity via dose-response curves. The resulting strains were compared with our previously reported Homo sapiens ERß biosensor strain. In initial tests, all three of the strains correctly identified estrogenic test compounds with a high degree of certainly (Z' typically greater than 0.5), including the weakly binding test compound bisphenol A (BPA) (Z' ≈ 0.1-0.3). The modular design of the sensing element in this strain allows quick development of new species-based biosensors by simple LBD swapping, suggesting its use in initial comparative analysis of EDC impacts across multiple species. Interestingly, the growth phenotypes of the biosensor strains indicate similar binding for highly estrogenic control compounds, but suggest differences in ligand binding for more weakly binding EDCs.
Asunto(s)
Técnicas Biosensibles , Disruptores Endocrinos/toxicidad , Escherichia coli/genética , Receptor beta de Estrógeno/genética , Peces Planos/metabolismo , Porcinos/metabolismo , Animales , Compuestos de Bencidrilo/toxicidad , Receptor beta de Estrógeno/agonistas , Receptor beta de Estrógeno/antagonistas & inhibidores , Peces Planos/genética , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Ligandos , Fenoles/toxicidad , Estructura Terciaria de Proteína , Especificidad de la Especie , Porcinos/genéticaRESUMEN
Glycoprotein therapeutics are currently used by large patient populations and generate significant revenue for the biopharmaceutical industry. These therapeutic proteins are currently purified at industrial scale using individualized processes involving multiple chromatographic steps. In the absence of a viable affinity platform method, the required chromatographic steps are difficult to develop and inevitably lead to significant yield losses. Further, during preclinical development, there is a need for reliable platform technologies capable of performing high-throughput screening for biologic candidates. Although affinity tags can provide a solution to some of these challenges, they require specific affinity resins, and the tag itself can interfere with the target protein characteristics. Fusion protein systems consisting of elastin-like polypeptide (ELP) and self-cleaving split inteins such as Npu DnaE can serve as potential non-chromatographic platform technologies for the single-step purification of tagless glycoproteins expressed in mammalian cells. In this chapter, we demonstrate the use of this technology to obtain highly purified anti-ErbB2 ML39 single-chain variable fragment (scFv) expressed from Expi293F suspension cells.
Asunto(s)
Inteínas , Anticuerpos de Cadena Única , Animales , Humanos , Elastina/genética , Cromatografía , Ensayos Analíticos de Alto Rendimiento , MamíferosRESUMEN
Protein splicing is a posttranslational process in which an intein segment excises itself from two flanking peptides, referred to as exteins. In the native context, protein splicing results in two separate protein products coupled to the activation of the intein-containing host protein. Inteins are generally described as either full-length inteins, mini-inteins or split inteins, which are differentiated by their genetic structure and features. Inteins can also be divided into three classes based on their splicing mechanisms, which differ in the location of conserved residues that mediate the splicing pathway. Although inteins were once thought to be selfish genetic elements, recent evidence suggests that inteins may confer a genetic advantage to their host cells through posttranslational regulation of their host proteins. Finally, the ability of modified inteins to splice and cleave their fused exteins has enabled many new applications in protein science and synthetic biology. In this review, we briefly cover the mechanisms of protein splicing, evidence for some inteins as environmental sensors, and intein-based applications in protein engineering.
RESUMEN
In this work, we describe a novel self-cleaving affinity tag technology based on a highly modified split-intein cleaving element. In this system, which has recently been commercialized by Protein Capture Science, LLC under the name iCapTagTM , the N-terminal segment of an engineered split intein is covalently immobilized onto a capture resin, while the smaller C-terminal intein segment is fused to the N-terminus of the desired target protein. The tagged target can then be expressed in an appropriate expression system, without concern for premature intein cleaving. During the purification, strong binding between the intein segments effectively captures the tagged target onto the capture resin while simultaneously generating a cleaving-competent intein complex. After unwanted impurities are washed from the resin, cleavage of the target protein is initiated by a shift of the buffer pH from 8.5 to 6.2. As a result, the highly purified tagless target protein is released from the column in the elution step. Alternately, the resin beads can be added directly to cell culture broth or lysate, allowing capture, purification and cleavage of the tagless target protein using a column-free format. These methods result in highly pure tagless target protein in a single step, and can thereby accelerate characterization and functional studies. In this work we demonstrate the single step purification of streptokinase, a fibrinolytic agent, and an engineered recombinant human hemoglobin 1.1 (rHb1.1). © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression of high-titer protein tagged with the Nostoc punctiforme (Npu) DnaE split-intein on the N-terminus Basic Protocol 2: Purification of high-titer protein using the Nostoc punctiforme (Npu) DnaE split-intein purification platform Alternate Protocol 1: Expression of low-titer protein tagged with the Nostoc punctiforme (Npu) DnaE split-intein on the N-terminus Alternate Protocol 2: Purification of low-titer protein using the Nostoc punctiforme (Npu) DnaE split-intein purification platform.
Asunto(s)
Inteínas , Nostoc , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Empalme de Proteína , Nostoc/genética , Nostoc/químicaRESUMEN
Thyroid receptor signaling controls major physiological processes and disrupted signaling can cause severe disorders that negatively impact human life. Consequently, methods to detect thyroid receptor ligands are of great toxicologic and pharmacologic importance. Previously, we reported thyroid receptor ligand detection with cell-free protein synthesis of a chimeric fusion protein composed of the human thyroid receptor beta (hTRß) receptor activator and a ß-lactamase reporter. Here, we report a 60% reduction in sensing cost by reengineering the chimeric fusion protein biosensor to include a reporter system composed of either the full-length beta galactosidase (ß-gal), the alpha fragment of ß-gal (ß-gal-α), or a split alpha fragment of the ß-gal (split ß-gal-α). These biosensor constructs are deployed using E. coli XL1-Blue cell extract to (1) avoid the ß-gal background activity abundant in BL21 cell extract and (2) facilitate ß-gal complementation reporter activity to detect human thyroid receptor ligands. These results constitute a promising platform for high throughput screening and potentially the portable detection of human thyroid receptor ligands.
RESUMEN
Self-cleaving affinity tags, based on engineered intein protein domains, have been touted as a universal single step purification platform for tagless non-mAb proteins. These approaches provide all of the power and flexibility of tag-based affinity methods, but deliver a tagless target protein suitable for clinical applications without complex process development. This combination of features might accelerate and de-risk biopharmaceutical development by bridging early discovery to full-scale manufacturing under a single platform. Despite this profound promise, intein-based technologies have yet to reach their full potential. This review examines the evolution of intein-based purification methods in the light of several significant intein patents filed over the last 3 decades. Illustrated with actual key figures from each of the relevant patents, key advances are described with a focus on applications in basic research and biopharmaceutical production. Suggestions for extending intein-based purification systems to emerging therapies and non-protein applications are presented as concluding remarks.
RESUMEN
Nuclear receptors (NRs) influence nearly every system of the body and our lives depend on correct NR signaling. Thus, a key environmental and pharmaceutical quest is to identify and detect chemicals which interact with nuclear hormone receptors, including endocrine disrupting chemicals (EDCs), therapeutic receptor modulators, and natural hormones. Previously reported biosensors of nuclear hormone receptor ligands facilitated rapid detection of NR ligands using cell-free protein synthesis (CFPS). In this work, the advantages of CFPS are further leveraged and combined with kinetic analysis, autoradiography, and western blot to elucidate the molecular mechanism of this biosensor. Additionally, mathematical simulations of enzyme kinetics are used to optimize the biosensor assay, ultimately lengthening its readable window by five-fold and improving sensor signal strength by two-fold. This approach enabled the creation of an on-demand thyroid hormone biosensor with an observable color-change readout. This mathematical and experimental approach provides insight for engineering rapid and field-deployable CFPS biosensors and promises to improve methods for detecting natural hormones, therapeutic receptor modulators, and EDCs.
Asunto(s)
Técnicas Biosensibles , Disruptores Endocrinos , Hormonas , Cinética , LigandosRESUMEN
In this work, we apply self-cleaving affinity tag technology to several target proteins secreted into the Escherichia coli periplasm, including two with disulfide bonds. The target proteins were genetically fused to a self-cleaving chitin-binding domain-intein tag for purification via a chitin-agarose affinity resin. By attaching the intein-tagged fusion genes to the PelB secretion leader sequence, the tagged target proteins were secreted to the periplasmic space and could be recovered in active form by simple osmotic shock. After chitin-affinity purification, the target proteins were released from the chitin-binding domain tag via intein self-cleaving. This was induced by a small change in pH from 8.5 to 6.5 at room temperature, allowing direct elution of the cleaved target protein from the chitin affinity resin. The target proteins include the E. coli maltose-binding protein and ß-lactamase enzyme, as well as two human antibody fragments that contain disulfide bonds. In all cases, the target proteins were purified with good activity and yield, without the need for refolding. Overall, this work demonstrates the compatibility of the ΔI-CM intein with the PelB secretion system in E. coli, greatly expanding its potential to more complex proteins.