Optimizing Escherichia coli as a protein expression platform to produce Mycobacterium tuberculosis immunogenic proteins
1 Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy
2 The Protein Factory, Interuniversity Centre Politecnico di Milano, ICRM CNR Milano and University of Insubria, Milan, Italy
3 Department of Drug Sciences and Italian Biocatalysis Centre, University of Pavia, Pavia, Italy
4 Department of Infection Diseases, Fondazione IRCCS-Policlinico San Matteo, Pavia, Italy
5 Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy
6 ProxAgen Ltd., Sofia, Bulgaria
7 Present address: Foundation Edmund Mach, San Michele all'Adige, Trento, Italy
Microbial Cell Factories 2013, 12:115 doi:10.1186/1475-2859-12-115Published: 19 November 2013
A number of valuable candidates as tuberculosis vaccine have been reported, some of which have already entered clinical trials. The new vaccines, especially subunit vaccines, need multiple administrations in order to maintain adequate life-long immune memory: this demands for high production levels and degree of purity.
In this study, TB10.4, Ag85B and a TB10.4-Ag85B chimeric protein (here-after referred as full) - immunodominant antigens of Mycobacterium tuberculosis - were expressed in Escherichia coli and purified to homogeneity. The rational design of expression constructs and optimization of fermentation and purification conditions allowed a marked increase in solubility and yield of the recombinant antigens. Indeed, scaling up of the process guaranteed mass production of all these three antigens (2.5-25 mg of pure protein/L cultivation broth). Quality of produced soluble proteins was evaluated both by mass spectrometry to assess the purity of final preparations, and by circular dichroism spectroscopy to ascertain the protein conformation. Immunological tests of the different protein products demonstrated that when TB10.4 was fused to Ag85B, the chimeric protein was more immunoreactive than either of the immunogenic protein alone.
We reached the goal of purifying large quantities of soluble antigens effective in generating immunological response against M. tuberculosis by a robust, controlled, scalable and economically feasible production process.