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Alternative approach to express Mycobacterium tuberculosis proteins in Escherichia coli.

the various aspects of gene expression in Escherichia coli has allowed geneticists not only to engineer increasingly sophisticated expression vectors to maximize gene expression but also to create a growing number of host strains for optimizing the expression of gene products. The advent of inducible expression systems, especially in the bacteriophage T7 RNA polymerase-based system (1–3), allows nearly routine overexpression at levels from 2% to as high as 50% of the cell protein. However, for unknown reasons, several proteins (usually of low molecular weight) are very difficult to express in E. coli host cells; typical examples are several proteins of Mycobacterium tuberculosis and Pseudomonas aeruginosa (4–7). The use of low copy number T7 expression vectors has improved the expression of some proteins of both microorganisms in E. coli (8). Another alternative to circumvent this constraint uses the standard artifice of engineering the genes encoding such proteins “fused” with genes encoding proteins that are readily overexpressed in E. coli. In addition, these proteins contain a specific binding motif to facilitate the purification and detection of the recombinant fusion proteins. Some common examples are calmodulin-binding peptide, glutathione-S-transferase (GST), maltose-binding protein, and thioredoxin (9). However, a serious limitation of this procedure is the obvious fact that the recombinant protein is “contaminated” with an unrelated molecule, which is a situation that is in direct opposition to the very concept of producing recombinant proteins to generate highly purified molecules. Despite the fact that most of these systems offer the option of specific digestion of the junction of the two fused proteins (by engineering specific enzymatic sites at the junction), followed by purification of the protein of interest, this procedure is cumbersome and often does not work properly. Moreover, many proteins are not expressed well even as fusion proteins. Here we report the results of a simple, alternative procedure to express M. tuberculosis problem proteins in E. coli. In contrast to engineering the genes of these proteins with a heterologous “carrier” gene, we constructed “homo-fusions” (i.e., fusions of two or three copies of the same gene in tandem expressed as dimers or trimers). This simple procedure resulted in the excellent expression of the immunologically reactive recombinant proteins. This alternative was initially used to express the gene of a small molecular weight protein of M. tuberculosis, which we could not otherwise express. The gene encoding this 6.4-kDa protein (which we named U2) [GenBank® accession no. CAB08507; open reading frame (ORF) Rv0909] has been under investigation in our laboratory as a potential diagnostics and vaccine candidate. Our initial approach, involving PCR amplification followed by cloning into the pET-17b vector and transformation into E. coli BL-21 (DE3) pLysS host cells (both from Novagen, Madison, WI, USA), was disappointing. Upon induction with IPTG, no significant expression of the protein could be obtained; in fact, a region of diffused band of lower molecular weight than expected was observed. No significant expression of this molecule could be achieved either with the BL-21(DE3) or the BL-21(DE3) pLysS. Before testing the expression of U2 in conventional and commercially available systems involving fusion proteins (hetero-fusions), we tested the possibility of facilitating expression by simply increasing the molecular weight of the protein using the artifice of constructing homofusions instead. Three sets of oligonucleotide PCR primers were used separately to amplify the full-length ORF of U2 from genomic DNA of the virulent Erdman strain of M. tuberculosis. Table 1 depicts the sequences of these three sets of primers. The resultant PCR products were double-digested with NdeI and HindIII, HindIII and BamHI, and BamHI and EcoRV, respectively. The three fragments were ligated (T4 DNA ligase; Stratagene, La Jolla, CA, USA) and cloned into the pET-17b vector in tandem, which was also double-digested with NdeI and HindIII, HindIII and BamHI, and BamHI and EcoRV, respectively, in successive rounds of digestion, ligation, transformation, and plasmid preparation. The result was three clones: one, the original copy of the BENCHMARKS