RESUMEN

A major obstacle to the use of whey protein in protein-enriched sports beverages is the heat-induced gelation of the protein in the presence of salt. In this study, whey protein soluble aggregates (WPSAs) with high tolerance to NaCl and heat were successfully generated by preheating whey protein isolate (WPI) at a low concentration (1 % w/v) and pH 8.5. The suspension of WPSAs (5 % w/v) with 100 mM NaCl maintained clarity, transparency, and good flowability even after 30 min of heating at 100 °C. However, suspensions prepared by untreated WPI turned into milky white gels. WPSAs had a reduced Zeta potential at pH 7 compared to WPI, making them more resistant to the electrostatic screening caused by NaCl. Additionally, WPSAs exhibited reduced sensitivity to heat treatment due to a more compact structure achieved through preheating modification. In light of these findings, a straightforward and effective method was presented for regulating the heat and ionic strength tolerance of whey protein aggregates.

Asunto(s)

Calor , Agregado de Proteínas , Proteína de Suero de Leche , Proteína de Suero de Leche/química , Concentración Osmolar , Concentración de Iones de Hidrógeno , Cloruro de Sodio/química , Manipulación de Alimentos/métodos

RESUMEN

The tendency of ovotransferrin (OVT) to unfold and aggregate under 60 °C severely restricted sterilization temperature during egg processing. Searching for efficient strategies to improve OVT thermal stability is essential for improving egg product quality and processing suitability. Here, we investigated the effect of sulfate polysaccharide (dextran sulfate, DS) on heat-induced aggregation of OVT. We found that DS can effectively suppress amorphous aggregation of OVT at pH 7.0 after heating. Strikingly, the addition of 5 µM DS fully suppressed insoluble aggregates formation of 0.5 mg/mL OVT. Structure analysis confirmed that DS preserves nearly the entire secondary and tertiary structure of OVT during heating. The steric hindrance effect arising from strong electrostatic interactions between OVT and DS, coupled with reduced OVT hydrophobicity, is the underlying mechanism in suppressing protein-protein interactions, thus enhancing thermal stability. These findings suggest DS could act as protein stabilizers and chaperones, enhancing the thermostability of heat-sensitive proteins.

Asunto(s)

Conalbúmina , Calor , Conalbúmina/química , Sulfato de Dextran , Temperatura , Electricidad Estática

RESUMEN

Soluble aggregates are reported to be the most neurotoxic species of α-Synuclein (αSyn) in Parkinson's disease (PD) and hence are a promising target for diagnosis and treatment of PD. However, the predominantly intracellular location of αSyn limits its accessibility, especially for antibody-based molecules and prompts the need for exceptionally strong soluble αSyn aggregate binders to enhance their sensitivity and efficacy for targeting the extracellular αSyn pool. In this study, we have created the multivalent antibodies TetraSynO2 and HexaSynO2, derived from the αSyn oligomer-specific antibody SynO2, to increase avidity binding to soluble αSyn aggregate species through more binding sites in close proximity. The multivalency was achieved through recombinant fusion of single-chain variable fragments of SynO2 to the antibodies' original N-termini. Our ELISA results indicated a 20-fold increased binding strength of the multivalent formats to αSyn aggregates, while binding to αSyn monomers and unspecific binding to amyloid ß protofibrils remained low. Kinetic analysis using LigandTracer revealed that only 80% of SynO2 bound bivalently to soluble αSyn aggregates, whereas the proportion of TetraSynO2 and HexaSynO2 binding bi- or multivalently to soluble αSyn aggregates was increased to ~ 95% and 100%, respectively. The overall improved binding strength of TetraSynO2 and HexaSynO2 implies great potential for immunotherapeutic and diagnostic applications with targets of limited accessibility, like extracellular αSyn aggregates. The ability of the multivalent antibodies to bind a wider range of αSyn aggregate species, which are not targetable by conventional bivalent antibodies, thus could allow for an earlier and more effective intervention in the progression of PD.

Asunto(s)

Enfermedad de Parkinson , alfa-Sinucleína , Humanos , Péptidos beta-Amiloides , Anticuerpos Monoclonales , Cinética

RESUMEN

Despite ongoing debate, the amyloid ß-protein (Aß) remains the prime therapeutic target for the treatment of Alzheimer's disease (AD). However, rational drug design has been hampered by a lack of knowledge about neuroactive Aß. To help address this deficit, we developed live-cell imaging of iPSC-derived human neurons (iNs) to study the effects of the most disease relevant form of Aß-oligomeric assemblies (oAß) extracted from AD brain. Of ten brains studied, extracts from nine caused neuritotoxicity, and in eight cases this was abrogated by Aß immunodepletion. Here we show that activity in this bioassay agrees relatively well with disruption of hippocampal long-term potentiation, a correlate of learning and memory, and that measurement of neurotoxic oAß can be obscured by more abundant non-toxic forms of Aß. These findings indicate that the development of novel Aß targeting therapeutics may benefit from unbiased activity-based discovery. To test this principle, we directly compared 5 clinical antibodies (aducanumab, bapineuzumab,  BAN2401, gantenerumab, and SAR228810) together with an in-house aggregate-preferring antibody (1C22) and established relative EC50s in protecting human neurons from human Aß. The results yielded objective numerical data on the potency of each antibody in neutralizing human oAß neuritotoxicity. Their relative efficacies in this morphological assay were paralleled by their functional ability to rescue oAß-induced inhibition of hippocampal synaptic plasticity. This novel paradigm provides an unbiased, all-human system for selecting candidate antibodies for advancement to human immunotherapy.

Asunto(s)

Enfermedad de Alzheimer , Humanos , Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Inmunoterapia , Neuronas/metabolismo

RESUMEN

Amyloid ß-protein (Aß) plays an initiating role in Alzheimer's disease (AD), but only a small number of groups have studied Aß extracted from human brain. Most prior studies have utilized synthetic Aß peptides, but the relevance of these test tube experiments to the conditions that prevail in AD is uncertain. Here, we describe three distinct methods for studying Aß from cortical tissue. Each method allows the analysis of different ranges of species thus enabling the examination of different questions. The first method allows the study of readily diffusible Aß with a relatively high specific activity. The second enables the analysis of readily solubilized forms of Aß the majority of which are inactive. The third details the isolation of true Aß dimers which have disease-related activity. We also describe a bioassay to study the effects of Aß on the neuritic integrity of iPSC-derived human neurons. The combined use of this bioassay and the described extraction procedures provides a platform to investigate the activity of different forms and mixtures of Aß species, and offers a tractable system to identify strategies to mitigate Aß mediated neurotoxicity.

RESUMEN

Solvent additives, including NaCl, arginine hydrochloride (ArgHCl), glycine and sucrose, are used to enhance protein stability or reduce protein aggregation. Here, we studied the effects of these additives on proteins using agarose native gel electrophoresis. Since these additives are used at relatively high concentration, we first confirmed that they do not interfere with the performance of the native gel electrophoresis. Agarose native gel electrophoresis showed that aggregation of bovine serum albumin (BSA) induced by heating was slightly reduced by NaCl and ArgHCl. On the contrary, glycine and sucrose had marginal effects. ArgHCl and NaCl promoted heat aggregation of monoclonal antibody (mAb), while glycine and sucrose stabilized the native mAb. Arginine methyl ester inhibited heat aggregation of lysozyme and, to a much lesser extent, BSA. These results show that agarose native gel electrophoresis can be used to analyze the effects of solvent additives on proteins subjected to heat stresses. SYPRO Orange that stains only unfolded proteins confirmed unfolded structures of soluble aggregates.

Asunto(s)

Muramidasa

RESUMEN

Formation of protein aggregates as inclusion bodies (IBs) still poses a major hurdle in the recovery of bioactive proteins from E. coli. Despite the development of many mild solubilization buffers in last two decades, high-throughput recovery of functional protein from wide range of IBs is still a challenge at an academic and industrial scale. Herein, a novel formulation for improved recovery of bioactive protein from variety of bacterial IBs is developed. This novel formulation is comprised of 20% trifluoroethanol, 20% n-propanol and 2 M urea at pH 12.5 which disrupts the major dominant forces involved in protein aggregation. An extensive comparative study of novel formulation conducted on different IBs demonstrates its high solubilization and refolding efficiency. The overall yield of bioactive protein from human growth hormone expressed as bacterial IBs is reported to be around 50%. This is attributed to the capability of novel formulation to disrupt the tertiary structure of the protein while protecting the secondary structure of the protein, thereby reducing the formation of soluble aggregates during refolding. Thus, the formulation can eliminate the need of screening and optimizing various solubilization formulation and will improve the efficiency of recovering bioactive protein from variety of IB aggregates.

Asunto(s)

Cuerpos de Inclusión/metabolismo , Proteínas/metabolismo , Escherichia coli/metabolismo , Hormona de Crecimiento Humana/metabolismo , Humanos , Replegamiento Proteico , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Solubilidad , Trifluoroetanol/metabolismo

RESUMEN

Protein aggregation likely plays a key role in the initiation and spreading of Alzheimer's disease pathology through the brain. Soluble aggregates of amyloid beta are believed to play a key role in this process. However, the aggregates present in humans are still poorly characterized due to a lack of suitable methods required for characterizing the low concentration of heterogeneous aggregates present. We have used a variety of biophysical methods to characterize the aggregates present in human Alzheimer's disease brains at Braak stage III. We find soluble amyloid beta-containing aggregates in all regions of the brain up to 200 nm in length, capable of causing an inflammatory response. Rather than aggregates spreading through the brain as disease progresses, it appears that aggregation occurs all over the brain and that different brain regions are at earlier or later stages of the same process, with the later stages causing increased inflammation.

RESUMEN

Myofibrillar proteins (MPs) have not been fully used for a long time due to its poor solubility in low ionic strength solutions. The study explored the effect of high pressure homogenization (HPH) cycles under two pressures on the solubility of MPs. The MPs solubility increased with HPH cycles (p < 0.05), the results of turbidity, appearance, droplet size indicated that the increase of solubility was due to MPs depolymerization, excessive HPH cycles (25k psi for 11 cycles) would lead to protein re-aggregation but does not affect solubility (p>0.05). SDS-PAGE suggested that myosin formed soluble polymers with different molecular weights through disulfide bonds during HPH cycles, the polymer consisted of myosin subunits of different molecular weights. Endogenous fluorescence spectra, intermolecular chemical forces, isoelectric point analysis and free amino acids (FAAs) indicated that the dissolution of polymers in low ionic strength media was dominated by polar environment and intermolecular steric hindrance, but not to FAAs.

Asunto(s)

Proteínas Musculares/química , Miofibrillas/química , Presión , Agregado de Proteínas , Aminoácidos/química , Animales , Concentración de Iones de Hidrógeno , Punto Isoeléctrico , Masculino , Microscopía de Fuerza Atómica , Nefelometría y Turbidimetría , Concentración Osmolar , Tamaño de la Partícula , Conejos , Solubilidad , Soluciones , Agua/química

RESUMEN

Unbalanced wine astringency, caused by a gap between phenolic and technological grape maturities, is one of the consequences of the global climate change in the vitiviniculture. To resolve it, potential strategies are being currently used, like the addition of commercial yeast mannoproteins (MPs) to wines. In this work, the main interactions responsible for the wine astringent sensation, namely, interactions between human salivary proteins and wine flavanols have been studied by Dynamic Light Scattering (DLS) and liquid chromatography coupled to DAD and MS detectors (HPLC-DAD-MS), in presence or absence of two MPs with different saccharide/protein ratio. The results indicate that there are differences on the substrate specificity for each mannoprotein and that its action mechanism could change not only depending on the mannoprotein composition but also on the flavanol structure. MPs with elevated carbohydrate content could act thought the stabilization of soluble aggregates with human salivary proteins and flavanols, mainly non-galloylated flavanol oligomers, whereas MPs with higher protein percentage mostly could precipitate flavanols (mainly non-galloylated ones with low degree of polymerization) which partially prevents the formation of insoluble flavanol-salivary protein aggregates.

Asunto(s)

Vino , Humanos , Glicoproteínas de Membrana , Polifenoles , Saccharomyces cerevisiae , Proteínas y Péptidos Salivales , Vino/análisis

RESUMEN

A solution (10%, w/v) of whey protein soluble aggregates (WPISA) was pretreated with high-intensity ultrasound (HUS, 20 kHz) for different durations (10-40 min) before incubation with transglutaminase (TGase) to investigate the effect of HUS on the structural, physicochemical, rheological, and gelation properties of TGase cross-linked WPISA. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that HUS increased the amounts of high-molecular-weight polymers/aggregates in WPISA after incubation with TGase. HUS significantly increased (P < 0.05) the degree of TGase-mediated cross-linking in WPISA, as demonstrated by a reduction in free amino group contents. HUS significantly increased (P < 0.05) the particle size, intrinsic fluorescence intensity, and surface hydrophobicity of TGase cross-linked WPISA, but had no significant impact (P > 0.05) on the zeta-potential or total free sulfhydryl group content of TGase cross-linked WPISA. The apparent viscosity and the consistency index of TGase cross-linked WPISA were significantly increased by HUS (P < 0.05), which indicated that HUS facilitated the formation of more high-molecular-weight polymers. HUS significantly increased (P < 0.05) the water holding capacity and gel strength of glucono-δ-lactone (GDL)-induced TGase cross-linked WPISA gels. The results indicated that HUS could be an efficient tool for modifying WPISA to improve its degree of TGase-mediated cross-linking, which would lead to improved rheological and gelation properties.

Asunto(s)

Fenómenos Químicos , Agregado de Proteínas , Reología , Transglutaminasas/metabolismo , Ondas Ultrasónicas , Geles , Solubilidad

RESUMEN

In this study, a novel method called selective proteolysis was applied to the glycinin component of soy protein isolate (SPI), and a degraded glycinin hydrolysate (DGH) was obtained. The effects of high-intensity ultrasound (HIU) treatment (20 kHz at 400 W, 0, 5, 20, and 40 min) on the physical, structural, and aggregation properties of DGH were investigated with the aim to reveal the influence of the selectively hydrolyzing glycinin component on the HIU treatment of soy protein. The effects of HIU on DGH and a control SPI (CSPI) were both time-dependent. HIU induced the formation of soluble aggregates in both samples at an early stage, while it dissociated these newly formed aggregates after a longer duration. Selectively hydrolyzing glycinin contributed to the soluble aggregation by exposing the compact protein structure and producing small protein fractions. The larger extent of hydrophobic interactions and disulfide bonds imparted a higher stability to the soluble protein aggregates formed in DGH. As a result, DGH displayed more ordered secondary structures, a higher solubility, and better gelling properties after the HIU treatment, especially at 20 min. The results of this study will be beneficial to the scientific community as well as industrial application.

RESUMEN

Subvisible aggregates of proteins are suspected to cause adverse immune response, and a recent FDA guideline has recommended the monitoring of micrometer-sized aggregates (2-10 µm) though recognizing that the underlying mechanism behind aggregation and immunogenicity remains unclear. Here, we report a correlation between the immunogenicity and the size of nanometer-scaled aggregates of a small 6.5 kDa model protein, bovine pancreatic trypsin inhibitor (BPTI) variant. BPTI-19A, a monomeric and nonimmunogenic protein, was oligomerized into subvisible aggregates with hydrodynamic radii (Rh) of 3-4 nm by attaching hydrophobic solubility controlling peptide (SCP) tags to its C-terminus. The results showed that the association of nonimmunogenic BPTI into nanometer-sized subvisible aggregates made it highly immunogenic, as assessed by the IgG antibody titers of the mice's sera. Overall, the study emphasizes that subvisible aggregates, as small as a few nanometers, which are presently ignored, are worth monitoring for deciphering the origin of undesired immunogenicity of therapeutic proteins.

Asunto(s)

Aprotinina/inmunología , Agregado de Proteínas/inmunología , Animales , Aprotinina/química , Femenino , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos ICR , Multimerización de Proteína , Solubilidad

RESUMEN

BACKGROUND: Fragile X-associated tremor/ataxia syndrome is a late-onset neurodegenerative disorder that affects about 40% of carriers of CGG-repeat expansions in the premutation range within the fragile X gene (FMR1). Main clinical features include intention tremor, cerebellar ataxia, and parkinsonism. Recently, great emphasis on the deposition of soluble aggregates produced by a RAN translation process, as main pathogenic mechanism, has been given. These aggregates contain a small protein with a polyglycine stretch on the aminoterminal end named FMRpolyG and, so far, have been isolated and characterized in drosophila and mouse models, in post mortem brain of fragile X-associated tremor/ataxia syndrome patients, in fibroblasts of fragile primary ovarian insufficiency patients, but never in fibroblasts from a fragile X-associated tremor/ataxia living patients. In adult carriers the syndrome is frequently misdiagnosed due to the lack of specific markers. METHODS: We standardized immunocytochemistry, immunoprecipitation and western blot procedures to study and biochemically characterize the FMRpolyG protein in fibroblasts from human skin biopsy. RESULTS: We demonstrate for the first time, in fibroblasts from a patient affected by Fragile X-associated tremor/ataxia syndrome, the presence ex vivo of inclusions consisting of FMRpolyG- Hsp70 soluble aggregates. CONCLUSION: These observations can pave the way to develop a cellular model for studying ex vivo and in vitro the mechanisms involved in the production of FMRpolyG aggregates, their toxicity, and the role of the FMRpolyG-Hsp70 interaction in the pathogenesis of fragile X-associated tremor/ataxia syndrome.

RESUMEN

Solubility is the prime criterion for determining the quality of recombinant proteins, yet it often fails to represent functional activity due to the involvement of non-functional, misfolded, soluble aggregates, which compromise the quality of recombinant proteins. However, guidelines for the quality assessment of soluble proteins have neither been proposed nor rigorously validated experimentally. Using the aggregation-prone enhanced green-fluorescent protein (EGFP) folding reporter system, we evaluated the folding status of recombinant proteins by employing the commonly used sonication and mild lysis of recombinant host cells. We showed that the differential screening of solubility and folding competence is crucial for improving the quality of recombinant proteins without sacrificing their yield. These results highlight the importance of screening out incorrectly folded soluble aggregates at the initial purification step to ensure the functional quality of recombinant proteins.

Asunto(s)

Agregado de Proteínas , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Cromatografía en Gel , Tamaño de la Partícula , Proteínas Recombinantes de Fusión , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Solubilidad , Sonicación

RESUMEN

Significant data suggest that soluble Aß oligomers play an important role in Alzheimer's disease (AD), but there is great confusion over what exactly constitutes an Aß oligomer and which oligomers are toxic. Most studies have utilized synthetic Aß peptides, but the relevance of these test tube experiments to the conditions that prevail in AD is uncertain. A few groups have studied Aß extracted from human brain, but they employed vigorous tissue homogenization which is likely to release insoluble Aß that was sequestered in plaques during life. Several studies have found such extracts to possess disease-relevant activity and considerable efforts are being made to purify and better understand the forms of Aß therein. Here, we compared the abundance of Aß in AD extracts prepared by traditional homogenization versus using a far gentler extraction, and assessed their bioactivity via real-time imaging of iPSC-derived human neurons plus the sensitive functional assay of long-term potentiation. Surprisingly, the amount of Aß retrieved by gentle extraction constituted only a small portion of that released by traditional homogenization, but this readily diffusible fraction retained all of the Aß-dependent neurotoxic activity. Thus, the bulk of Aß extractable from AD brain was innocuous, and only the small portion that was aqueously diffusible caused toxicity. This unexpected finding predicts that generic anti-oligomer therapies, including Aß antibodies now in trials, may be bound up by the large pool of inactive oligomers, whereas agents that specifically target the small pool of diffusible, bioactive Aß would be more useful. Furthermore, our results indicate that efforts to purify and target toxic Aß must employ assays of disease-relevant activity. The approaches described here should enable these efforts, and may assist the study of other disease-associated aggregation-prone proteins.

Asunto(s)

Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Fragmentos de Péptidos/metabolismo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/líquido cefalorraquídeo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Células CHO , Cricetulus , Femenino , Humanos , Potenciación a Largo Plazo/efectos de los fármacos , Potenciación a Largo Plazo/genética , Potenciación a Largo Plazo/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Peso Molecular , Mutación/genética , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Fragmentos de Péptidos/farmacología , Células Madre Pluripotentes/metabolismo

RESUMEN

This Personal Account describes collaborative investigations into apocryphal microwave effects in organic chemistry. Focused research on microwave-assisted organic synthesis has been fraught with confusion, controversy, and misinformation. Microwave heating is an undoubtedly useful tactic for organic synthesis, but whether or not it can offer strategic advantages remains an open question in the minds of many people. (Ironically, those who do not consider it an open question are split as to whether it has been resolved affirmatively or negatively.) Our research in this area is guided by the hypothesis that microwave heating can alter reaction kinetics in ways distinct from what is observable under conventional heating. Here we provide a succinct record of the origins of our interests, our initial queries and associated controversies, and recent efforts to identify, quantify, and begin to leverage selective microwave heating for strategic advantage in organic synthesis.

RESUMEN

Molecular and chemical chaperones /foldases can strongly contribute to improve the amounts and the structural quality of recombinant proteins. Several methodologies have been proposed to optimize their beneficial effects. This chapter presents a condensed summary of the biotechnological opportunities offered by this approach followed by a protocol describing the method we use for expressing disulfide bond-dependent recombinant antibodies in the cytoplasm of bacteria engineered to overexpress sulfhydryl oxidase and DsbC isomerase. The system is based on the possibility to trigger the foldase expression independently and before the induction of the target protein. As a consequence, the recombinant antibody synthesis starts only after enough foldases have accumulated to promote correct folding of the antibody.

Asunto(s)

Anticuerpos/genética , Clonación Molecular/métodos , Escherichia coli/genética , Ingeniería Genética/métodos , Pliegue de Proteína , Animales , Anticuerpos/química , Anticuerpos/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Agregado de Proteínas , Proteína Disulfuro Isomerasas/genética , Proteína Disulfuro Isomerasas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Solubilidad , Regulación hacia Arriba

RESUMEN

Morpholino oligomers are effective antisense molecules to regulate gene expression and the US FDA has approved a Morpholino drug for the treatment of Duchenne muscular dystrophy. However, it has been observed that the antisense activities of aqueous solutions of some Morpholinos decrease over time. We hypothesize that the decreased activity is caused by the formation of soluble aggregates of the Morpholinos. Here, we analyzed three Morpholino sequences by size exclusion chromatography and found two of them have over time formed soluble aggregates in water. The degree of aggregation is sequence-, temperature-, and time-dependent. We describe a simple procedure for detecting and breaking down the aggregates to return the Morpholinos to their monomeric forms.

Asunto(s)

Morfolinos/química , Morfolinos/genética , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/genética , Emparejamiento Base , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Solubilidad , Soluciones

RESUMEN

Maltose-Binding Protein (MBP) is one of the most popular fusion partners being used for producing recombinant proteins in bacterial cells. MBP allows the use of a simple capture affinity step on Amylose-Agarose or Dextrin-Sepharose columns, resulting in a protein that is often 70-90 % pure in a single step. In addition to protein isolation applications, MBP provides a high degree of translation, and facilitates the proper folding and solubility of the target protein. This paper describes efficient procedures for isolating highly purified MBP target proteins. Special attention is given to considerations for downstream applications such as structural determination studies, protein activity assays, and assessing the chemical characteristics of the target protein.

Asunto(s)

Cromatografía de Afinidad/métodos , Cromatografía en Gel/métodos , Proteínas de Unión a Maltosa , Proteínas Recombinantes de Fusión/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Proteínas de Unión a Maltosa/química , Proteínas de Unión a Maltosa/genética , Agregado de Proteínas , Pliegue de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Solubilidad