A novel bicistronic vector for overexpressing Mycobacterium tuberculosis proteins in Escherichia coli

Abstract

A putative DNA glycosylase encoded by the Rv3297 gene (MtuNei2) has been identified in Mycobacterium tuberculosis. Our efforts to express this gene in Escherichia coli either by supplementing tRNAs for rare codons or optimizing the gene with preferred codons for E. coli resulted in little or no expression. On the other hand, high-level expression was observed using a bicistronic expression vector in which the target gene was translationally coupled to an upstream leader sequence. Further comparison of the predicted mRNA secondary structures supported the hypothesis that mRNA secondary structure(s) surrounding the translation initiation region (TIR), rather than codon usage, played the dominant role in influencing translation efficiency, although manipulation of codon usage or tRNA supplementation did further enhance expression in the bicistronic vector. Addition of a cleavable N-terminal tag also facilitated gene expression in E. coli, possibly through a similar mechanism. However, since cleavage of N-terminal tags is determined by the amino acid at the P(1)' position downstream of the protease recognition sequence and results in the addition of an extra amino acid in front of the N-terminus of the protein, this strategy is not particularly amenable to Fpg/Nei family DNA glycosylases which carry the catalytic proline residue at the P(1)' position and require a free N-terminus. On the other hand, the bicistronic vector constructed here is potentially valuable particularly when expressing proteins from G/C rich organisms and when the proteins carry proline residues at the N-terminus in their native form. Thus the bicistronic expression system can be used to improve translation efficiency of mRNAs and achieve high-level expression of mycobacterial genes in E. coli.

Fig. 1

Expression of Rv3297 (MtuNei2) in the pET30a vector in the presence of pRARE2 tRNA plasmid. Uninduced (lanes 1, 3 and 5) and induced (lanes 2, 4 and 6) whole cell lysates of transformed BL21-Gold (DE3) cells were analyzed on a 12% SDS–PAGE gel. Lane M, Novagen Perfect Protein^™ Marker (15–150 kDa). Lane P, purified MtuNei2-His (29.6 kDa).

Fig. 2

Schematic of the bicistronic vectors used in this study. (a) Parental pET30a-ORF6 vector, (b) Rv3297 cloned into the pET30a-ORF6. RBS for the upstream leader sequence and the downstream target gene are boxed. The cloning sites for the Rv3297 (MtuNei2) gene are underlined in the parental vector. Nucleotides ‘AT’ of the EcoRV recognition sequence overlap with the stop codon for the upstream ORF6 and the initiation codon for the downstream target gene.

Fig. 3

Induction of Rv3297 (MtuNei2) expression in pET30a and the pET30a-ORF6 bicistronic vector in the presence of pRARE tRNA plasmid or pACYC184 missing the tRNA genes. Uninduced (lanes 1, 3, 5, 7 and 9) and induced (lanes 2, 4, 6, 8 and 10) whole cell lysates of transformed BL21-Gold (DE3) cells were analyzed on a 12% SDS–PAGE. Lane M, Novagen Perfect Protein^™ Marker (15–150 kDa). Lane P, purified MtuNei2-His (29.6 kDa).

Fig. 4

Partial mRNA conformations of the first 165 nucleotides transcribed in pET30a and the pET30a-ORF6 constructs. (a) pET30a/cRv3297-His, (b) pET30a/sRv3297-His, (c) pET30a-ORF6/cRv3297-His, (d) pET30a-ORF6/sRv3297-His. Only secondary structures near the RBS (shaded) and AUG start codons (bold) are shown. The free energy (ΔG) values for the individual stem-loop structures (aside) and for the folded 165 nt mRNA (below) are shown.

Fig. 5

Expression of cloned Rv3297 (MtuNei2) as N- or C-terminal hexa-his tagged fusion proteins. Uninduced (lanes 1, 3, 5, 7 and 9) and induced (lanes 2, 4, 6, 8 and 10) whole cell lysates of transformed BL21-Gold (DE3) cells were analyzed on a 12% SDS–PAGE. Lane M, Novagen Perfect Protein^™ Marker (15–150 kDa). Lane P, purified MtuNei2-His (29.6 kDa).

Fig. 6

Induction of sRv2464 (MtuNei1) and cRv2924 (MtuFpg1) expression in the pET30a-ORF6 bicistronic vector. Uninduced (lanes 1 and 3) and induced (lanes 2 and 4) whole cell lysates of transformed BL21-Gold (DE3) cells were analyzed on a 12% SDS–PAGE. Lane M, Novagen Perfect Protein^™ Marker (15–150 kDa). Lane P1, purified MtuNei1-His (30.8 kDa). Lane P2, purified MtuFpg1-His (33.0 kDa).