title: A versatile bacterial expression vector designed for single-step cloning of multiple DNA fragments using homologous recombination.
abstract: Production of recombinant proteins is the starting point for biochemical and biophysical analyses and requires methodology to efficiently proceed from gene sequence to purified protein. While optimized strategies for the efficient cloning of single-gene fragments for bacterial expression is available, efficient multiple DNA fragment cloning still presents a challenge. To facilitate this step, we have developed an efficient cloning strategy based on yeast homologous recombination cloning (YHRC) into the new pET-based bacterial expression vector pSUMO-YHRC. The vector supports cloning for untagged expression as well as fusions to His6-SUMO or His6 tags. We demonstrate that YHRC from single PCR products of 6 independent genes into the vector results in virtually no background. Importantly, in a quantitative assay for functional expression we find that single-step YHRC of 7 DNA fragments can be performed with very high cloning efficiencies. The method and reagents described in this paper significantly simplifies the construction of expression plasmids from multiple DNA fragments, including complex gene fusions, chimeric genes and polycistronic constructs.
authors.affiliation: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, S-10691 Stockholm, Sweden.
authors.forename: Mats A
authors.initials: MA
authors.lastname: Holmberg
authors.affiliation: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, S-10691 Stockholm, Sweden.
authors.forename: Naveen Kumar Chandappa
authors.initials: NK
authors.lastname: Gowda
authors.affiliation: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, S-10691 Stockholm, Sweden. Electronic address: claes.andreasson@su.se.
authors.forename: Claes
authors.initials: C
authors.lastname: Andréasson
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D059765:Homologous Recombination; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins
keywords: Cloning; Nanobody; Protein expression; Protein purification; Recombination cloning SUMO