Signal peptide prediction suggests Mycobacterium tuberculosis curli pilin subunit secretion via the Sec pathway may hinder MTP overexpression in Escherichia coli

Introduction Mycobacterium tuberculosis curli pili (MTP) are novel, potential TB diagnostic biomarkers as they are important virulence attributes, unique to the M. tuberculosis complex (MTBC). The production of high quality recombinant transmembrane and secretory proteins that can serve as biomarkers may be challenging due to their secretion attributes. For example, the signal peptide of MTP governed by the classical secretion pathway may hinder the purification of the protein in E. coli systems. In this study, the secretion characteristics of MTP were determined and the cloning, expression and purification of MTP was attempted in E.coli. Materials and methods A fragment of MTP unique to MTBC was cloned into pet101 and pGEX-6P-1 vectors. The clones were confirmed by nucleotide sequencing and expression of the protein was attempted at IPTG concentrations ranging from 0.1mM to 1mM and at temperatures between 25 °C to 37 °C. The pGEX-6P-1/mtp clone expressed protein was purified, yielding a MTP-GST fusion protein and a free GST band that were analysed by LC/MS mass spectrometry. Inclusion body preparation attempted from the pet101/mtp clone yielded two bands at 10 kDa and below 10 kDa, both of which were analysed by LC/MS mass spectrometry. Transcription activity of both the clones was interrogated by real time PCR on the cDNA derived from the induced clones at various time points after induction with IPTG. The signal peptide and protein secretion characteristics of the MTP protein were determined by bioinformatics analysis of the amino acid sequence using publically available software. Results The truncated MTP fragments were successfully cloned in both the vectors as confirmed by nucleotide sequencing. Expression of the pGEX-6P-1/mtp clone using 0.5 mM IPTG at 27 °C demonstrated the presence of the expected fragment at approximately 35 kDa. This was confirmed by Western Blotting using anti-GST antibodies. However, purification of MTP in adequate quantities as a pure protein fraction was unsuccessful. Mass spectrometry did not detect any fragments belonging to MTP, but only those of E.coli membrane proteins for the pet101/mtp clone and fragments of the GST tag in the case of the pGEX-6P-1/mtp clone. The bioinformatics secretion analyses of MTP predicted a strong Sec regulated secretion pathway and the absence of non-classical “mycobacterial specific” secretion. Discussion M. tuberculosis membrane and secretory proteins often contain signal peptides. In this study, excluding the signal peptide region and using a GST tag greatly enhanced the expression of the protein in the soluble fraction. However, purification of the MTP peptide remained problematic due to a lower available peptide concentration resulting from the lower molecular weight, in the purified fraction compared to the GST tag. Alternately, the predicted Sec regulated secretion pathway may play a role in inhibition of MTP overexpression in E.coli. Thus, alternatives to E. coli expression systems or more efficient purification strategies are required for the acquisition of high quality M. tuberculosis antigens.