title: Construction of two Lactococcus lactis expression vectors combining the Gateway and the NIsin Controlled Expression systems.
abstract: Over the last 10 years, the NIsin Controlled Expression (NICE) system has been extensively used in the food-grade bacterium Lactococcus lactis subsp. cremoris to produce homologous and heterologous proteins for academic and biotechnological purposes. Although various L. lactis molecular tools have been developed, no expression vectors harboring the popular Gateway recombination system are currently available for this widely used cloning host. In this study, we constructed two expression vectors that combine the NICE and the Gateway recombination systems and we tested their applicability by recombining and over-expressing genes encoding structural proteins of lactococcal phages Tuc2009 and TP901-1. Over-expressed phage proteins were analyzed by immunoblotting and purified by His-tag affinity chromatography with protein productions yielding 2.8-3.7 mg/l of culture. This therefore is the first description of L. lactis NICE expression vectors which integrate the Gateway cloning technology and which are suitable for the production of sufficient amounts of proteins to facilitate subsequent structural and functional analyses.
authors.affiliation: Department of Microbiology, University College Cork, Western Road, Cork, Ireland. f.douillard@ucc.ie
authors.forename: François P
authors.initials: FP
authors.lastname: Douillard
authors.forename: Jennifer
authors.initials: J
authors.lastname: Mahony
authors.forename: Valérie
authors.initials: V
authors.lastname: Campanacci
authors.forename: Christian
authors.initials: C
authors.lastname: Cambillau
authors.forename: Douwe
authors.initials: D
authors.lastname: van Sinderen
mesh_terms: D001435:Bacteriophages / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics*; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism / Q000821:virology; D009561:Nisin / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D009561:Nisin