Improved expression vector for Brucella species.

We constructed an improved vector pNSGroE for gene expression studies in Brucella spp. It is derived from the broad host range cloning vector pBBR1MCS (1). This new plasmid has several advantages over pBBR1MCS or its derivatives pBBGroE (2): (i) it is smaller in size, 2.9 kb; (ii) it expresses proteins as His-tagged fusion for easy detection and purification; (iii) it carries the groE promoter for constitutive expression that is enhanced under conditions of stress in vitro and in vivo; and (iv) it has higher levels of heterologous protein expression. Our expression studies in Brucella abortus strain RB51 indicated that the level of heterologous protein expression is higher with pNSGroE compared to pBBGroE vector. We have also demonstrated the ability of the new vector to express heterologous fusion proteins stably in Brucella species. The smaller size of the new vector was achieved by combining only the very essential elements for replication and expression [minimum origin of replication, promoter, antibiotic selection marker, and multiple cloning sites (MCS)] and removing all nonessential elements in the original vector pBBR1MCS or pBBGroE (lacZ gene, mobilization gene, and 1 kb upstream of the groE promoter). The vector was constructed by excision of a 185bp fragment containing the MCS of pRSETA vector (Invitrogen, Carlsbad, CA, USA) using XbaI and HindIII. This fragment was cloned into XbaI and HindIII sites of pGEM11 vector (Promega, Madison, WI, USA) to form construct A. The groE promoter of Brucella spp. was amplified from the genomic DNA of B. abortus strain 2308 using Platinum® PCR SuperMix High Fidelity kit (Invitrogen). In the reverse primer (Table 1), six histidine and one glycine residues were engineered after the translational start codon to facilitate the epitope tagging at the amino terminus of any expressed protein. The amplified groE promoter was cloned into BamHI and SalI sites of construct A to form construct B. The rep gene necessary for the replication of pBBR1 plasmid (3) was amplified along with its own promoter from the pBBR1MCS vector using a primer pair with SalI and XbaI sites. The chloramphenicol resistance gene (cat), along with its own promoter, was amplified from the pBBR1MCS using a primer pair with SpeI and HindIII sites. An XbaI site was engineered into the reverse primer of the cat gene to add an extra unique restriction site within the MCS of the new plasmid followed by a transcriptional stop codon. The groE promoter along with the downstream MCS was excised from construct B using SalI and HindIII sites. The rep gene was digested with SalI and XbaI, and the cat gene product was digested with SpeI and HindIII. The three fragments were purified and ligated to form plasmid pNSGroE (Figure 1) (GenBank® accession no. AY576605). Strain RB51 was transformed by electroporation according Improved expression vector for Brucella species