High-level heterologous gene expression in Ochrobactrum anthropi using an A-rich UP element

Abstract

DNA regions that flank a gene's promoter play an important role in determining transcription efficiency by interacting with the carboxy-terminal domain of RNA polymerase alpha-subunit. We placed an adenine-rich upstream element (UP) between -38 and -59 of the core trc promoter to enhance gene expression in Ochrobactrum anthropi up to 66-fold. The high level of expression achieved by the UP element and the N-terminus fusion of a 6xHis epitope tag facilitated detection and purification of heterologous proteins directly from O. anthropi.

Vectors for enhanced gene expression and protein purification in Salmonella

“…There was no change in activity using A+T rich trcB promoter. This enhancement appears to be due to ability of the 'A tract' to provide binding site(s) for the RNA polymerase that contributes to the wrapping of the promoter DNA around the RNA polymerase (Aiyar et al, 1998;Seleem et al, 2007a).…”

“…We placed an adenine (A)-rich and A+T rich upstream element (UP) (Estrem et al, 1998) between −40 and −59 bp of the core trc promoter (Seleem et al, 2007a). The 'A tract' sequence (TrcD) had a positive effect on trc promoter activity in S. Choleraesuis, when positioned at −40 bp, increasing transcription 40% (Fig.1B) over the core trc promoter.…”

Simple method for transformation of Ochrobactrum anthropi

“…Recently the ability of O. anthropi to express recombinant fusion proteins was revealed in one-step detection and purification of recombinant green fluorescence protein (gfp) without using E. coli as an expression host (Seleem et al 2007). We were able to obtain enough electrocompetent cells (2.5 9 10 10 cells/ml) and (3 9 10 11 cells/ml) from liquid culture and plate method, respectively, to perform 80 (2 ml of 25 ll reaction) and 120 (3 ml of 25 ll reaction) transformation reactions with efficiency 2.1 9 10 9 and 9.5 9 10 6 transformants per microgram of DNA, respectively.…”

“…To date, genetic manipulation of Ochrobactrum spp. has been limited due to the inefficiency of DNA transformation and the lack of an efficient and stable replicating vector (Seleem et al 2007). Transformation of O. anthropi using the two methods described for related species Agrobacterium tumefaciens and Rhizobium leguminosarum (Holsters et al 1978) were not successful (unpublished data).…”

Development of a dual vaccine for prevention of Brucella abortus infection and Escherichia coli O157:H7 intestinal colonization

“…1A, the lacZ promoter present in the vector pBBR1MCS-4 was replaced by the stronger promoter Trc. An A-rich UP element described for the improved heterologous expression of proteins in Ocrobactrum arthropii [33] was added upstream to improve antigen expression in Brucella (Fig. 1A).…”