Improved Expression Vector for Brucella Species

Abstract

We constructed an improved vector pNSGroE for gene expression stud-ies 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 car-ries the groE promoter for constitutive expression that is enhanced under con-ditions 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 pNS-GroE compared to pBBGroE vector. We have also demonstrated the ability of the new vector to express heterolo-gous fusion proteins stably in Brucella species. The smaller size of the new vector was achieved by combining only the very essential elements for replica-tion and expression [minimum origin of replication, promoter, antibiotic selection marker, and multiple cloning sites (MCS)] and removing all nones-sential 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 185-bp 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