Optimizing Escherichia coli as a protein expression platform to produce Mycobacterium tuberculosis immunogenic proteins

Abstract

BackgroundA 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.ResultsIn 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.ConclusionsWe 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.

Cloning and expression of acyl homoserine lactone (AHL) lactonase genes of Bacillus cereus INT1c and Bacillus thuringiensis SGT3g in Escherichia coli

“…Expression of recombinant AHL-lactonase genes also can reduce virulence of E. carotovora on potatoes (Lee et al, 2002). Expression of recombinant proteins is influenced by various factors such as time of induction, long incubation after induction, incubation temperature, type of vectors and bacterial hosts (Dang et al, 2012;Fang et al, 2014;Picaud et al, 2007;Piubelli et al, 2013). Conclusively, AHL-lactonase genes of B. cereus INT1c and B. thuringiensis SGT3g are expressed in E. coli BL21 cells (DE3) pLysS.…”

Bioprocess development workflow: Transferable physiological knowledge instead of technological correlations

“…This approach will aid to extend individual product-specific findings to strain-specific platform knowledge. 14…”

“…In this work, we investigate the same strain background as in the work of Wechselberger et al with a different product. This approach will aid to extend individual product‐specific findings to strain‐specific platform knowledge …”

Rational design, preparation and characterization of recombinant Ag85B variants and their glycoconjugates with T-cell antigenic activity againstMycobacterium tuberculosis

“…28 Thus, different saccharides activated with the 2-iminomethoxyethyl (IME) reactive linker were used for conjugation with this MTB protein, allowing the preparation of the corresponding glycoconjugates. 29,30 By combining the analytical characterization of glycosylation sites with the biological evaluation of neoglycoproteins, the effect of the glycosylation on rAg85B antigenic activity was investigated.…”

“…28 Briey, E. coli BL21(DE3) cells transformed with the pET32b-Ag85B plasmids encoding for the different protein variants were grown in SB medium at 37 C,…”