One‐plasmid tunable coexpression for mycobacterial protein–protein interaction studies

Abstract

A single plasmid that allows controlled coexpression has been developed for use in mycobacteria. The tetracycline inducible promoter, PtetO, was used to provide tetracyclinedependent induction of one gene, while the Psmyc, Pimyc, or Phsp promoters were used to provide three different levels of constitutive expression of a second gene. The functions of these four individual promoters were established using green fluorescent protein (GFP) and a newly identified red fluorescence inducible protein from Geobacillus sterothermophilus strain G1.13 (RFIP) as reporters. The tandem use of GFP and RFIP as reporter genes allowed optimization of the tunable coexpression in Mycobacterium smegmatis; either time at a fixed inducer concentration or changes in inducer concentration could be used to control the protein:protein ratio. This single vector system was used to coexpress the two-protein Mycobacterium tuberculosis stearoyl-CoA D 9 desaturase complex (integral membrane desaturase Rv3229c and NADPH oxidoreductase Rv3230c) in M. smegmatis. The catalytic activity was found to increase in a manner corresponding to increasing the level of Rv3230c relative to a fixed level of Rv3229c. This system, which can yield finely tuned coexpression of the fatty acid desaturase complex in mycobacteria, may be useful for study of other multicomponent complexes. Furthermore, the tunable coexpression strategy used herein should also be applicable in other species with minor modifications.

Regulated Expression Systems for Mycobacteria and Their Applications

“…TetON and TetOFF have been used by several groups to analyze gene functions in M. smegmatis and M. tuberculosis (Table 1). They also provide the basis for some of the other regulatory expression systems developed more recently (22–24) and a tunable coexpression system to analyze protein-protein interactions (25). …”

To catch a killer. What can mycobacterial models teach us about Mycobacterium tuberculosis pathogenesis?

“…Moreover, the essential nature of the mycobacterial proteosome during growth of M. tuberculosis on agar, in liquid media, and in mice was characterized with this system [6••]. In another creative application of the Tet-inducible system, a plasmid was constructed that allows for tunable expression of multiple mycobacterial proteins in vivo [9•] (Figure 1). Both the Tet-ON, Tet-OFF and one-plasmid expression vectors were initially optimized in M. smegmatis and/or BCG and then applied to M. tuberculosis [7,9•].…”

“…Right, tunable expression of GFP in an atc dose-dependent manner, compared to constitutive expression of RFIP. From [9] with permission.…”

PSI:Biology-materials repository: a biologist’s resource for protein expression plasmids

“…These well-tested vectors provide a resource for the research community to quickly and easily express their proteins of interest. In particular, vector development has focused on optimization of protein expression in bacterial (29–31), wheat germ cell-free (32), yeast (33), or mycobacterial (34) systems. PSI centers have also focused on designing protein expression vectors to overcome common obstacles in protein expression pipelines by increasing protein solubility (35), improving enzymatic activity by co-expressing proteins that are required for full catalytic activity (36), or decreasing protein toxicity (37).…”