title: Optimizing Escherichia coli as a protein expression platform to produce Mycobacterium tuberculosis immunogenic proteins.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy. flavia.marinelli@uninsubria.it.
authors.forename: Luciano
authors.initials: L
authors.lastname: Piubelli
authors.forename: Manuela
authors.initials: M
authors.lastname: Campa
authors.forename: Caterina
authors.initials: C
authors.lastname: Temporini
authors.forename: Elisa
authors.initials: E
authors.lastname: Binda
authors.forename: Francesca
authors.initials: F
authors.lastname: Mangione
authors.forename: Massimo
authors.initials: M
authors.lastname: Amicosante
authors.forename: Marco
authors.initials: M
authors.lastname: Terreni
authors.forename: Flavia
authors.initials: F
authors.lastname: Marinelli
authors.forename: Loredano
authors.initials: L
authors.lastname: Pollegioni
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D046528:Chimerism; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology*; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000276:immunology*
chemical_list: D001426:Bacterial Proteins