title: Use of Strep-tag II for rapid detection and purification of Mycobacterium tuberculosis recombinant antigens secreted by Streptomyces lividans.
abstract: 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.
authors.affiliation: Institute of Pharmacy and Food, University of Havana, Havana, Cuba. yul@uab.edu
authors.forename: Julio C
authors.initials: JC
authors.lastname: Ayala
authors.forename: Elsa
authors.initials: E
authors.lastname: Pimienta
authors.forename: Caridad
authors.initials: C
authors.lastname: Rodríguez
authors.forename: Jozef
authors.initials: J
authors.lastname: Anné
authors.forename: Carlos
authors.initials: C
authors.lastname: Vallín
authors.forename: María T
authors.initials: MT
authors.lastname: Milanés
authors.forename: Emmanuel
authors.initials: E
authors.lastname: King-Batsios
authors.forename: Kris
authors.initials: K
authors.lastname: Huygen
authors.forename: Lieve
authors.initials: L
authors.lastname: Van Mellaert
mesh_terms: D000217:Acyltransferases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D000907:Antibodies, Bacterial / Q000097:blood / Q000276:immunology; D000942:Antigens, Bacterial / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D001426:Bacterial Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000235:genetics; D009842:Oligopeptides / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D018709:Statistics, Nonparametric; D048170:Streptomyces lividans / Q000378:metabolism*; D014376:Tuberculosis / Q000276:immunology
chemical_list: C099517:Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly; D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D000217:Acyltransferases; C112580:antigen 85A, Mycobacterium tuberculosis
keywords: Affinity chromatography; Mycobacterium tuberculosis; Purification; Secretory protein production; Strep-tag® II; Streptomyces lividans