Asunto(s)
Basigina/química , Secuencia de Aminoácidos , Basigina/genética , Cristalografía por Rayos X , Dimerización , Humanos , Modelos Moleculares , Peso Molecular , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
Bone morphogenetic protein-7 (BMP-7, OP-1) is a secreted growth factor that is predominantly known for its osteoinductive properties, though it has also been implicated as having a role in mammalian kidney development. Clinical efficacy of recombinant BMP-7 has been demonstrated in the treatment of orthopedic injuries through topical application. However, the pharmaceutical development of recombinant BMP-7 for systemic delivery has presented many challenges. Specifically, the expression level of recombinant mature BMP-7 protein in mammalian cells is very low, the molecule has poor solubility at neutral pH, and intracellular proteolytic processing events result in a secreted BMP-7 having multiple amino-termini, creating a heterogeneous mixture of proteins. Utilizing structural information, we have designed and generated a number of rational BMP-7 mutations that improved both expression levels in mammalian cells and solubility at neutral pH, while limiting the amino-terminal heterogeneity of the mature protein. Introduction of these mutations did not compromise BMP-7 in vitro bioactivity. This improved BMP-7 molecule is better suited for pharmaceutical development and clinical advancement for indications where systemic delivery may be required.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Mutantes/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Fosfatasa Alcalina/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Proteína Morfogenética Ósea 7 , Proteínas Morfogenéticas Óseas/química , Proteínas Morfogenéticas Óseas/genética , Células CHO , Línea Celular , Cricetinae , Cricetulus , Dimerización , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes/biosíntesis , Proteínas Mutantes/genética , Mutación/genética , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Ratas , Factor de Crecimiento Transformador beta/química , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Cytokines in plasmid form can act as potent adjuvants when co-administered with DNA vaccines, resulting in an enhanced immune response to the DNA-encoded antigen. This is true of interleukin-18 (IL-18), which has been shown to serve as an adjuvant in conjunction with certain DNA vaccines. To determine if the properties of IL-18 could be optimized for use as a DNA vaccine adjuvant, a model of IL-18/IL-18R binding was developed to identify variants of human IL-18 that were predicted to improve receptor interactions and potentially bioactivity. The linkage of mature IL-18 to a secretion signal sequence provided improved protein expression from mammalian cells and signal peptidase cleavage of this protein produced the authentic N-terminus. The IL-18 variant proteins secreted this way were bioactive, as demonstrated by their ability to induce interferon gamma (IFNgamma) expression by human peripheral blood mononuclear cells (PBMCs) and to bind to IL-18R, as demonstrated by BIAcore analysis. The IL-18 variants were inhibited by IL-18 binding protein (IL-18BP), the soluble inhibitor of IL-18, as measured by neutralization of the IFNgamma response in PBMCs. One variant, V11I/T63A, demonstrated increases both in bioactivity and mammalian cell expression as compared to native IL-18, indicating that this molecule may be particularly well suited for use as a DNA-encoded vaccine adjuvant.