RESUMO
B lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor superfamily of cytokines. When the 152 amino acids of the C-terminus are associated into a homotrimer, this protein exhibits the ability to stimulate B cell proliferation and differentiation. Since numerous potential therapeutic indications have been identified for BLyS and other BLyS-derived products, large quantities of the protein are needed to further basic research and clinical trials. In this work, we have developed a high yield recombinant expression system that utilizes Escherichia coli as the host organism. Recombinant soluble BLyS (rsBLyS) production was achieved through the use of the phoA promoter system. This expression system, coupled to a semi-defined fermentation process, resulted in final purified yields of 435 mg/L of properly folded, trimeric, biologically active rsBLyS. This level of production is an 11-fold increase in volumetric yields compared to the process currently being used for clinical production. Furthermore, the increased rsBLyS production obtained from this process enabled the development of a conventional purification scheme that eliminated the use of a BLyS-affinity resin.
Assuntos
Proteínas de Membrana/biossíntese , Proteínas de Membrana/isolamento & purificação , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/isolamento & purificação , Sequência de Aminoácidos , Animais , Fator Ativador de Células B , Linfócitos B/fisiologia , Reatores Biológicos/microbiologia , Western Blotting , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Códon , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Fermentação , Genes Bacterianos , Vetores Genéticos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/farmacologia , Camundongos , Dados de Sequência Molecular , Plasmídeos , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Solubilidade , Transformação Genética , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Anthrax is caused by the gram-positive spore-forming bacterium Bacillus anthracis. The anthrax toxin consists of three proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF). PA facilitates the translocation of LF and EF into the cytosol of mammalian cells. LF is thought to be a zinc-dependent metalloprotease that results in death. EF is a calmodulin- and calcium-dependent adenylate cyclase that causes edema upon entrance into the cytosol by elevating the cAMP levels in cells. Previous efforts to produce recombinant EF (rEF) in Escherichia coli yielded only 2.5 mg of rEF per liter of culture. In this work, we produced soluble rEF in large quantities in both the periplasm and cytoplasm of E. coli from shake flasks and fermentors. The rEF protein was purified by standard chromatography and yielded >97% pure, biologically active rEF. Yields of purified rEF from medium cell density fermentations resulted in up to 2.38 g/L of highly pure, biologically active rEF protein. These results exhibit the ability to generate gram quantities of active rEF from E. coli.
Assuntos
Adenilil Ciclases/biossíntese , Adenilil Ciclases/química , Bacillus anthracis/metabolismo , Escherichia coli/metabolismo , Engenharia de Proteínas/métodos , Adenilil Ciclases/genética , Adenilil Ciclases/isolamento & purificação , Adenilil Ciclases/farmacologia , Sequência de Aminoácidos , Animais , Antígenos de Bactérias , Bacillus anthracis/genética , Toxinas Bacterianas , Células CHO , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Escherichia coli/genética , Dados de Sequência Molecular , Peso Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Transformação BacterianaRESUMO
Anthrax is caused by the gram-positive, spore-forming bacterium, Bacillus anthracis. The anthrax toxin consists of three proteins, protective antigen (PA), lethal factor, and edema factor. Current vaccines against anthrax use PA as their primary component since it confers protective immunity. In this work, we expressed soluble, recombinant PA in relatively high amounts in the periplasm of E. coli from shake flasks and bioreactors. The PA protein was purified using Q-Sepharose-HP and hydroxyapatite chromatography, and routinely found to be 96-98% pure. Yields of purified PA varied depending on the method of production; however, medium cell density fermentations resulted in approximately 370 mg/L of highly pure biologically active PA protein. These results exhibit the ability to generate gram quantities of PA from E. coli.
