RESUMO
Insect cell-baculovirus expression vector system is one of the most established platforms to produce biological products, and it plays a fundamental role in the context of COVID-19 emergency, providing recombinant proteins for treatment, diagnosis, and prevention. SARS-CoV-2 infection is mediated by the interaction of the spike glycoprotein trimer via its receptor-binding domain (RBD) with the host's cellular receptor. As RBD is required for many applications, in the context of pandemic it is important to meet the challenge of producing a high amount of recombinant RBD (rRBD). For this reason, in the present study, we developed a process based on Sf9 insect cells to improve rRBD yield. rRBD was recovered from the supernatant of infected cells and easily purified by metal ion affinity chromatography, with a yield of 82% and purity higher than 95%. Expressed under a novel chimeric promoter (polh-pSeL), the yield of rRBD after purification was 21.1 ± 3.7 mg/L, which is the highest performance described in Sf9 cell lines. Finally, rRBD was successfully used in an assay to detect specific antibodies in COVID-19 serum samples. The efficient strategy herein described has the potential to produce high-quality rRBD in Sf9 cell line for diagnostic purpose.
Assuntos
COVID-19 , SARS-CoV-2 , Animais , SARS-CoV-2/genética , Baculoviridae/genética , Ligação Proteica , Insetos , Glicoproteína da Espícula de CoronavírusRESUMO
Serology testing for COVID-19 is important in evaluating active immune response against SARS-CoV-2, studying the antibody kinetics, and monitoring reinfections with genetic variants and new virus strains, in particular, the duration of antibodies in virus-exposed individuals and vaccine-mediated immunity. In this study, recombinant S protein of SARS-CoV-2 was expressed in Rachiplusia nu, an important agronomic plague. One gram of insect larvae produces an amount of S protein sufficient for 150 determinations in the ELISA method herein developed. We established a rapid production process for SARS-CoV-2 S protein that showed immunoreactivity for anti-SARS-CoV-2 antibodies and was used as a single antigen for developing the ELISA method with high sensitivity (96.2%) and specificity (98.8%). Our findings provide an efficient and cost-effective platform for large-scale S protein production, and the scale-up is linear, thus avoiding the use of complex equipment like bioreactors.
Assuntos
Teste Sorológico para COVID-19 , COVID-19/diagnóstico , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/biossíntese , Animais , Larva/metabolismo , Larva/virologia , Nucleopoliedrovírus , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , SARS-CoV-2/metabolismo , Células Sf9 , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , SpodopteraRESUMO
In this work, the influence of Sodium Acetate Trihydrate (SAT) on the gelling stage of a chitin hydrogel was studied. Characterization techniques, such as FTIR, Raman, solid-state NMR, Dielectric Spectroscopy, Small-angle X-ray scattering (SAXS), Wide-angle X-ray scattering (WAXS), and X-ray diffraction (XRD) were used to study the effect of SAT on the micro and nanostructure of the material in the wet, dry and freeze-dried states. It was demonstrated that the amount of SAT in the gelling solution can induce a variation in the supramolecular interaction among the polysaccharide chains, which leads to a change in the structural characteristics. In addition, it was observed that the polymer-water interactions are also altered by this structural ordering. Also, the affinity interaction with lysozyme was evaluated and an influence on the adsorption capacity was evidenced with the use of SAT. This could be an advance for biotechnological, biomedical, and food applications.
Assuntos
Quitina/química , Géis/química , Acetato de Sódio/química , Acetatos/química , Adsorção , Coloides , Liofilização/métodos , Espectroscopia de Ressonância Magnética/métodos , Muramidase/metabolismo , Nanoestruturas/química , Espalhamento a Baixo Ângulo , Difração de Raios X/métodosRESUMO
Feline interferon beta is a cytokine that belongs to the type I IFN family, with antitumor, antiviral and immunomodulatory functions. In this work, recombinant feline interferon beta (rFeIFNß) was expressed in insect larvae that constitute important agronomic plagues. rFeIFNß accumulated in the hemolymph of Spodoptera frugiperda larvae infected with recombinant baculovirus and was purified by Blue-Sepharose chromatography directly from larval homogenates on day 4 post-infection. rFeIFNß was recovered after purification with a specific activity of 1 × 106 IU mg-1. By this method, we obtained 8.9 × 104 IU of purified rFeIFNß per larva. The product was biologically active in vitro, with an antiviral activity of 9.5 × 104 IU mL-1, as well as a potent antitumor activity comparable to that of the commercial FeIFNω. The glycosylation of rFeIFNß was confirmed by peptide-N-glycosidase F digestion. Our findings provide a cost-effective platform for large-scale rFeIFNß production in laboratory research or veterinary medicine applications.
RESUMO
A cation exchange matrix with zwitterionic and multimodal properties was synthesized by a simple reaction sequence coupling sulfanilic acid to a chitosan based support. The novel chromatographic matrix was physico-chemically characterized by ss-NMR and ζ potential, and its chromatographic performance was evaluated for lysozyme purification from diluted egg white. The maximum adsorption capacity, calculated according to Langmuir adsorption isotherm, was 50.07 ± 1.47 mg g-1 while the dissociation constant was 0.074 ± 0.012 mg mL-1 . The process for lysozyme purification from egg white was optimized, with 81.9% yield and a purity degree of 86.5%, according to RP-HPLC analysis. This work shows novel possible applications of chitosan based materials. The simple synthesis reactions combined with the simple mode of use of the chitosan matrix represents a novel method to purify proteins from raw starting materials. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:387-396, 2018.
Assuntos
Quitosana/química , Clara de Ovo/química , Muramidase/isolamento & purificação , Ácidos Sulfanílicos/química , Adsorção , Soluções Tampão , Cromatografia Líquida de Alta Pressão , Espectroscopia de Ressonância Magnética , Muramidase/metabolismo , Concentração OsmolarRESUMO
Casein glycomacropeptide (CMP) is a 64- amino acid peptide found in cheese whey, which is released after κ-casein specific cleavage by chymosin. CMP lacks aromatic amino acids, a characteristic that makes it usable as a nutritional supplement for people with phenylketonuria. CMP consists of two nonglycosylated isoforms (aCMP A and aCMP B) and its different glycosylated forms (gCMP A and gCMP B). The most predominant carbohydrate of gCMP is N-acetylneuraminic acid (sialic acid). Here, we developed a CMP purification process based on the affinity of sialic acid for wheat germ agglutinin (WGA). After formation of chitosan beads and adsorption of WGA, the agglutinin was covalently attached with glutaraldehyde. Two matrices with different WGA density were assayed for CMP adsorption. Maximum adsorption capacities were calculated according to the Langmuir model from adsorption isotherms developed at pH 7.0, being 137.0 mg/g for the matrix with the best performance. In CMP reduction from whey, maximum removal percentage was 79% (specifically 33.7% of gCMP A and B, 75.8% of aCMP A, and 93.9% of aCMP B). The CMP was recovered as an aggregate with an overall yield of 64%. Therefore, the matrices developed are promising for CMP purification from cheese whey. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:171-180, 2017.
Assuntos
Aminoácidos/química , Caseínas/isolamento & purificação , Ácido N-Acetilneuramínico/química , Fragmentos de Peptídeos/isolamento & purificação , Proteínas do Soro do Leite/isolamento & purificação , Adsorção , Aminoácidos/metabolismo , Animais , Caseínas/química , Bovinos , Quitosana/química , Cromatografia de Afinidade , Glicosilação , Leite/química , Fragmentos de Peptídeos/química , Soro do Leite/química , Proteínas do Soro do Leite/químicaRESUMO
The worldwide production of whey increases by around 186 million tons each year and it is generally considered as a waste, even when several whey proteins have important economic relevance. For its valorization, inexpensive ligands and integrated chromatography methods need to be developed for specific and low-cost protein purification. Here, we describe a novel affinity process with the dye Yellow HE-4R immobilized on Sepharose for bovine lactoferrin purification. This approach based on a low-cost ligand showed an efficient performance for the recovery and purification of bovine lactoferrin directly from whey, with a yield of 71% and a purification factor of 61.
Assuntos
Cromatografia de Afinidade/métodos , Lactoferrina/isolamento & purificação , Proteínas do Leite/isolamento & purificação , Animais , Bovinos , Cromatografia de Afinidade/instrumentação , Corantes/química , Lactoferrina/química , Ligantes , Proteínas do Leite/química , Proteínas do Soro do LeiteRESUMO
Colloidal gold is the first choice for labeling antibodies to be used in Point Of Care Testing. However, there are some recent reports on a family of textile dyes-named "reactive dyes"-being suitable for protein labeling. In the present article, protein labeling conditions were optimized for Remazol Brilliant Violet 5R, and the sensitivity of the labeled antibodies was assessed and compared with that of colloidal-gold labeled antibodies. Also, the accelerated stability was explored. Optimal conditions were pH 10.95, dye:Ab molar ratio of 264 and an incubation time of 132 min. Labeled antibodies were stable, and could be successfully used in a slot blot assay, detecting as low as 400 ng/mL. Therefore, the present work demonstrates that vinylsulphonic reactive dyes can be successfully used to label antibodies, and are excellent candidates for the construction of a new generation of Point of Care Testing kits.
Assuntos
Anticorpos/análise , Corantes , Naftalenossulfonatos , Coloração e Rotulagem/métodos , Anticorpos/química , Proteínas Sanguíneas/análise , Coloide de Ouro , Humanos , Imunoglobulina G/análise , Naftalenossulfonatos/química , Sistemas Automatizados de Assistência Junto ao Leito , Estabilidade ProteicaRESUMO
Bovine viral diarrhea virus (BVDV) is considered an important cause of economic loss within bovine herds worldwide. In Argentina, only the use of inactivated vaccines is allowed, however, the efficacy of inactivated BVDV vaccines is variable due to its low immunogenicity. The use of recombinant subunit vaccines has been proposed as an alternative to overcome this difficulty. Different studies on protection against BVDV infection have focused the E2 protein, supporting its putative use in subunit vaccines. Utilization of transgenic plants expressing recombinant antigens for the formulation of experimental vaccines represents an innovative and cost effective alternative to the classical fermentation systems. The aim of this work was to develop transgenic alfalfa plants (Medicago sativa, L.) expressing a truncated version of the structural protein E2 from BVDV fused to a molecule named APCH, that target to antigen presenting cells (APCH-tE2). The concentration of recombinant APCH-tE2 in alfalfa leaves was 1 µg/g at fresh weight and its expression remained stable after vegetative propagation. A methodology based an aqueous two phases system was standardized for concentration and partial purification of APCH-tE2 from alfalfa. Guinea pigs parentally immunized with leaf extracts developed high titers of neutralizing antibodies. In bovine, the APCH-tE2 subunit vaccine was able to induce BVDV-specific neutralizing antibodies. After challenge, bovines inoculated with 3 µg of APCH-tE2 produced in alfalfa transgenic plants showed complete virological protection.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/prevenção & controle , Vírus da Diarreia Viral Bovina Tipo 1/imunologia , Medicago sativa/genética , Medicago sativa/imunologia , Vacinas Virais/farmacologia , Animais , Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Doença das Mucosas por Vírus da Diarreia Viral Bovina/imunologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Bovinos , Vírus da Diarreia Viral Bovina Tipo 1/genética , Feminino , Cobaias , Masculino , Plantas Geneticamente Modificadas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Resultado do Tratamento , Vacinação/veterinária , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/farmacologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Vacinas Virais/genética , Vacinas Virais/imunologiaRESUMO
An efficient affinity chromatographic matrix based on chitosan for wheat germ agglutinin (WGA) purification was developed. The matrices assayed consisted of chitosan mini-spheres cross-linked with epichlorhydrin 45, 250 or 500 mM. The maximum adsorption capacity of pure WGA - calculated from the corresponding isotherms - was between 43.2 and 48.9 mg/g at pH 5.0 and between 16.6 and 27.6 mg/g at pH 8.5. However, the adsorption of agglutinin from wheat germ extract was higher at pH 8.5. In addition, 0.5 g of mini-spheres cross-linked with epichlorhydrin 250 mM adsorbed 94.5% of the WGA present in 5 mL of the concentrated extract. Acetic acid was able to elute 100% of the adsorbed WGA. The purity of the WGA obtained was greater than 95% and the purification factor was 56.8. The matrix was able to maintain an efficient performance of the purification process for three consecutive cycles. A new method to monitor the purification process by RP-HPLC was developed.