RESUMO
A novel strategy for in vivo immobilization of enzymes on the surfaces of inclusion bodies has been established. It relies on expression in Escherichia coli of the polyhydroxybutyrate synthase PhaC from Cupriavidus necator, which carries at its amino terminus an engineered negatively charged alpha-helical coil (Ecoil) and forms inclusion bodies upon high-level expression. Coexpression in the same cell of galactose oxidase (GOase) from Fusarium spp. carrying a carboxy-terminal positively charged coil (lysine-rich coil [Kcoil]) sequence results in heterodimeric coiled-coil formation in vivo and in the capture of the enzyme in active form on the surface of the inclusion body particle. These round-shaped enzyme-decorated microparticles, with sizes of approximately 0.7 mum, can be isolated from lysed cells simply by centrifugation. The cost-effective one-step generation and isolation of enzymes immobilized on inclusion body particles may become useful for various applications in bioprocessing and biotransformation.
Assuntos
Aciltransferases/biossíntese , Cupriavidus necator/enzimologia , Enzimas Imobilizadas/isolamento & purificação , Escherichia coli/genética , Fusarium/enzimologia , Galactose Oxidase/biossíntese , Corpos de Inclusão/enzimologia , Aciltransferases/genética , Cupriavidus necator/genética , Enzimas Imobilizadas/biossíntese , Escherichia coli/enzimologia , Fusarium/genética , Galactose Oxidase/genética , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
A novel method for stepwise in vitro affinity maturation of antigen-specific shark vNAR domains is described that exclusively relies on semi-synthetic repertoires derived from non-immunized sharks. Target-specific molecules were selected from a CDR3-randomized bamboo shark (Chiloscyllium plagiosum) vNAR library using yeast surface display as platform technology. Various antigen-binding vNAR domains were easily isolated by screening against several therapeutically relevant antigens, including the epithelial cell adhesion molecule (EpCAM), the Ephrin type-A receptor 2 (EphA2), and the human serine protease HTRA1. Affinity maturation was demonstrated for EpCAM and HTRA1 by diversifying CDR1 of target-enriched populations which allowed for the rapid selection of nanomolar binders. EpCAM-specific vNAR molecules were produced as soluble proteins and more extensively characterized via thermal shift assays and biolayer interferometry. Essentially, we demonstrate that high-affinity binders can be generated in vitro without largely compromising the desirable high thermostability of the vNAR scaffold.
Assuntos
Afinidade de Anticorpos , Antígenos de Neoplasias/imunologia , Moléculas de Adesão Celular/imunologia , Imunoglobulinas/metabolismo , Receptor EphA2/química , Receptores de Antígenos/metabolismo , Serina Endopeptidases/química , Animais , Especificidade de Anticorpos , Antígenos de Neoplasias/química , Autoantígenos/química , Autoantígenos/metabolismo , Proteínas de Transporte , Moléculas de Adesão Celular/química , Molécula de Adesão da Célula Epitelial , Serina Peptidase 1 de Requerimento de Alta Temperatura A , Humanos , Imunoglobulinas/química , Imunoglobulinas/imunologia , Técnicas In Vitro , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Biblioteca de Peptídeos , Receptor EphA2/imunologia , Receptores de Antígenos/química , Receptores de Antígenos/imunologia , Serina Endopeptidases/imunologia , Tubarões/imunologiaRESUMO
A method for the detection of haloalkane conversion to the corresponding alcohols by haloalkane dehalogenases is described. It is based on a multistage enzyme reaction which allows for the analysis of alkyl halides in buffered systems. Irreversible hydrolytic dehalogenation catalyzed by haloalkane dehalogenase DhaA from Rhodococcus erythropolis transfers an alkyl halide into a corresponding alcohol that is further oxidized by alcohol oxidase AOX from Pichia pastoris yielding a respective aldehyde and hydrogen peroxide easily detectable via the horseradish peroxidase catalyzed oxidation of chromogenic molecules. Due to its high sensitivity (0.025 mM, 0.43 ppm for 1,3-dibromopropane), low expenditure and the ability of handling a large number of samples in parallel, this method is an attractive alternative to existing procedures for the monitoring of both haloalkanes and dehalogenases.