RESUMO
l-asparaginase catalyzes the conversion of l-asparagine to l-aspartate and ammonium. This protein is an important therapeutic enzyme used for the treatment of acute lymphoblastic leukemia. In this study, the asparaginase II-encoding gene ASP3 from Saccharomyces cerevisiae was cloned into the expression vector pET28a in-fusion with a 6x histidine tag and was expressed in Escherichia coli BL21 (DE3) cells. The protein was expressed at a high level (225.6 IU/g cells) as an intracellular and soluble molecule and was purified from the supernatant by nickel affinity chromatography. The enzyme showed very low activity against l-glutamine. The denaturing electrophoresis analysis indicated that the recombinant protein had a molecular mass of â¼38â¯kDa. The native enzyme was a tetramer with a molecular mass of approximately 178â¯kDa. The enzyme preparation showed antitumor activity against the K562 and Jurkat cell lines comparable or even superior to the E. coli commercial asparaginase.
Assuntos
Antineoplásicos/metabolismo , Asparaginase/genética , Proteínas de Bactérias/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , Antineoplásicos/química , Asparaginase/química , Asparaginase/metabolismo , Asparagina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Linhagem Celular Tumoral , Clonagem Molecular , Expressão Gênica , Glutamina/metabolismo , Humanos , Peso Molecular , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
With the recent outbreaks of Zika and Dengue virus infections in various countries worldwide, production of vaccines or diagnostic kits is an urgent public health demand. Production of a monoclonal antibody (mAb) that specifically binds to a common antigen shared by the Flavivirus genus will be necessary for new diagnostic kits or characterization and viral identity tests during vaccine development. This study aimed to cultivate, in serum-free conditions, the 4G2 hybridoma that produces an mAb, which recognizes a shared epitope from the Flavivirus genus. We compared 4G2 hybridoma growth and biochemical profiles between cells cultivated in batch mode over 10 days in roller bottles containing Dulbecco's modified Eagle's medium high glucose containing 10% fetal bovine serum medium or hybridomas directly adapted to Ex-Cell serum-free medium. Cellular parameters such as specific growth rate (µ), maximum cell concentration, specific l-lactate, and glucose and IgG rates were evaluated. Thereafter, we also compared total mAb volumetric productivity, purification yield, and mAb staining of Vero cells infected with Zika and Dengue-2 virus. Direct adaptation to serum-free conditions did not change hybridoma growth rate and mAb production under the conditions tested. Instead, serum-free mAb purification showed a higher yield with no alterations on mAb structure or mAb staining of Zika and Dengue Vero-infected cells.
Assuntos
Anticorpos Monoclonais/imunologia , Hibridomas/citologia , Zika virus/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Técnicas de Cultura Celular por Lotes/instrumentação , Técnicas de Cultura Celular por Lotes/métodos , Chlorocebus aethiops , Meios de Cultura Livres de Soro , Eletroforese em Gel de Poliacrilamida , Epitopos , Flavivirus/imunologia , Camundongos Endogâmicos BALB C , Células VeroRESUMO
PURPOSE: Acinetobacter baumannii is an important opportunistic pathogen that causes pneumoniae, urinary tract infections, and/or septicemia in immunocompromised patients. This pathogen is frequently associated with nosocomial outbreaks worldwide and has become particularly problematic because of its prevalence and resistance patterns to several antibiotics. In the present study, we used an immunoproteome-based approach to identify immunogenic proteins located on the surface of A. baumannii for the development of a possible immunotherapy against this devastating bacterial infection. EXPERIMENTAL DESIGN: Sera from patients with A. baumannii infections (n = 50) and from a control group of healthy individuals (n = 3) were analyzed for reactivity against A. baumannii outer membrane proteins (OMPs) using Western blot analysis. To identify potential immunogenic proteins in A. baumannii, OMPs were separated by 2DE, and reactive sera from infected patients were randomly selected and divided into two different pools, each containing 15 sera. Finally, MALDI-TOF/TOF mass spectrometric analysis was employed to identify the corresponding proteins. RESULTS: This analysis identified six immunoreactive proteins: OmpA, Omp34kDa, OprC, OprB-like, OXA-23, and ferric siderophore receptor protein. Notably, these proteins are highly abundant on the bacterial surface and involved in virulence, antibiotic resistance, and growth. CONCLUSIONS AND CLINICAL RELEVANCE: Our results support the notion that the proteins identified in the present immunoproteome study could serve as antigen candidates for the development of vaccines and passive immunotherapies against A. baumannii infections.