Streptomyces as a host for the secretion of heterologous proteins for the production of biopharmaceuticals

The commercial production of therapeutic or diagnostic proteins in recombinant microorganisms is of considerable interest. Several microbial protein production systems have been developed. So far, Escherichia coli have been the commonly employed host. However, proteins expressed in this host remain intracellular and often precipitate as inclusion bodies, which may seriously complicate downstreamprocessing. Faced with this problem, several genera of Gram-positive bacteria are being tested as host for the production of heterologous proteins due to their ability to efficiently secrete proteins in the culture medium. Among them is the genus Streptomyces since several of its species are known to secrete high amounts of proteins. Due to the absence of an extensive restriction-modification system, limited protease activity and the availability of suitable vector systems, Streptomyces lividans is the host of choice for the secretory production of heterologous proteins. The presented results show, that S. lividans can act as an interesting host to produce a number of proteins useful in several disease areas important in the worldwide pharmaceutical sales: i.e. oncology, immunology, cardiovascular diseases and infectious diseases

Recombinant production of Streptococcus equisimilis streptokinase by Streptomyces lividans

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Use of Strep-tag II for rapid detection and purification of Mycobacterium tuberculosis recombinant antigens secreted by Streptomyces lividans

Recent results with respect to the secretory production of bio-active Mycobacterium tuberculosis proteins in Streptomyces have stimulated the further exploitation of this host as a bacterial cell factory. However, the rapid isolation of a recombinant protein by conventional procedures can be a restrictive step. A previous attempt to isolate recombinant antigens fused to the widely used 6His-tag was found to be relatively incompatible with secretory production in the Streptomyces host. As an alternative, the eight-residue Strep-tag® II (WSHPQFEK), displaying intrinsic binding affinity towards streptavidin, was evaluated for the secretory production of two M. tuberculosis immunodominant antigens in Streptomyces lividans and their subsequent downstream processing. Therefore, the genes ag85A (Rv3804c, encoding the mycolyl-transferase Ag85A) and Rv2626c (encoding hypoxic response protein 1), were equipped with a 3'-Strep-tag® II-encoding sequence and placed under control of the Streptomyces venezuelae CBS762.70 subtilisin inhibitor (vsi) transcriptional, translational and signal sequences. Strep-tagged Ag85A and Rv2626c proteins were detected in the spent medium of recombinant S. lividans cultures at 48h of growth, and purified using a Strep-Tactin Superflow® matrix. Recombinant Ag85A appeared as a 30-kDa protein of which the N-terminal amino acid sequence was identical to the expected one. Rv2626c was produced in two forms of 17 and 37kDa respectively, both with the same predicted N-terminal sequence, suggesting that the 37-kDa product is an Rv2626c dimer. The obtained results indicate that the Strep-tagII is proteolytically stable in Streptomyces and does not interfere with the membrane translocation of Ag85A and Rv2626c. A comparison of reactivity of serum from tuberculosis patients versus healthy persons by ELISA showed that both S. lividans-derived antigens were recognized by sera of individuals infected with M. tuberculosis, indicating that they remained antigenetically active. To our knowledge, this is the first report showing the usefulness of an affinity peptide for detection and efficient downstream processing of recombinant proteins produced in Streptomyces. The present results add up strength to the significance of S. lividans as a valuable host to produce M. tuberculosis proteins with vaccine and diagnostic potential.status: publishe

La literatura popular impresa en España y en la América Colonial. Formas y temas, géneros, funciones, difusión, historia y teoría

El libro, publicado bajo la dirección del prof. Pedro M. Cátedra, contiene más de treinta monografías sobre la literatura popular impresa de los siglos XV al XVIII en España y América, con acercamientos muy variados y desde las perspectivas de la historia de la cultura popular, la historia de la imprenta, la aculturación tipográfica, los generos editoriales, etc., etc