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

Piubelli L, Campa M, Temporini C, Binda E, Mangione F, Amicosante M, Terreni M, Marinelli F, Pollegioni L - Microb. Cell Fact. (2013)

Bottom Line: 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).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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy. flavia.marinelli@uninsubria.it.

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.

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CD spectra of TB10.4 (continuous line), Ag85B (dashed line) and His-full2 (dotted line). A) Far-UV CD spectra (protein concentration: 0.1 mg/mL). B) Near-UV CD spectra (protein concentrations: 0.45, 0.35 and 0.85 mg/mL for TB10.4, Ag85B and His-full2, respectively). All spectra were collected in 10 mM ammonium acetate, pH 7, at 15°C.
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Figure 6: CD spectra of TB10.4 (continuous line), Ag85B (dashed line) and His-full2 (dotted line). A) Far-UV CD spectra (protein concentration: 0.1 mg/mL). B) Near-UV CD spectra (protein concentrations: 0.45, 0.35 and 0.85 mg/mL for TB10.4, Ag85B and His-full2, respectively). All spectra were collected in 10 mM ammonium acetate, pH 7, at 15°C.

Mentions: The quality of the recombinant antigenic proteins was assessed in terms of purity by SDS-PAGE (Additional file 2: Figure S7) and MS analyses (see above) and in terms of protein conformation by circular dichroism (CD) spectroscopy. The secondary structure of pure Ag85B (as determined by the far-UV CD spectra reported in Figure 6A) is composed of both α-helices and β-sheets (ca. 26 and 23% as estimated by k2D2 software vs. 37 and 20% from the crystal structure, pdb code 1F0N), while for TB10.4 the signal for α-helices only is apparent, in good agreement with the known 3D structure (55% α-helix content, pdb code 1F0N) and previous analyses [12]. Similarly, the signal for the tertiary structure significantly differs for the two M. tuberculosis antigens, see Figure 6B. The near- and far-UV spectra of His-full2 resemble those of Ag85B, according to the fact that Ag85B molecular mass is 3-fold higher than that of TB10.4 (Table 1). All together, spectral analyses indicate the acquisition of a well defined protein conformation for all the recombinant soluble antigenic proteins.


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

Piubelli L, Campa M, Temporini C, Binda E, Mangione F, Amicosante M, Terreni M, Marinelli F, Pollegioni L - Microb. Cell Fact. (2013)

CD spectra of TB10.4 (continuous line), Ag85B (dashed line) and His-full2 (dotted line). A) Far-UV CD spectra (protein concentration: 0.1 mg/mL). B) Near-UV CD spectra (protein concentrations: 0.45, 0.35 and 0.85 mg/mL for TB10.4, Ag85B and His-full2, respectively). All spectra were collected in 10 mM ammonium acetate, pH 7, at 15°C.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4225511&req=5

Figure 6: CD spectra of TB10.4 (continuous line), Ag85B (dashed line) and His-full2 (dotted line). A) Far-UV CD spectra (protein concentration: 0.1 mg/mL). B) Near-UV CD spectra (protein concentrations: 0.45, 0.35 and 0.85 mg/mL for TB10.4, Ag85B and His-full2, respectively). All spectra were collected in 10 mM ammonium acetate, pH 7, at 15°C.
Mentions: The quality of the recombinant antigenic proteins was assessed in terms of purity by SDS-PAGE (Additional file 2: Figure S7) and MS analyses (see above) and in terms of protein conformation by circular dichroism (CD) spectroscopy. The secondary structure of pure Ag85B (as determined by the far-UV CD spectra reported in Figure 6A) is composed of both α-helices and β-sheets (ca. 26 and 23% as estimated by k2D2 software vs. 37 and 20% from the crystal structure, pdb code 1F0N), while for TB10.4 the signal for α-helices only is apparent, in good agreement with the known 3D structure (55% α-helix content, pdb code 1F0N) and previous analyses [12]. Similarly, the signal for the tertiary structure significantly differs for the two M. tuberculosis antigens, see Figure 6B. The near- and far-UV spectra of His-full2 resemble those of Ag85B, according to the fact that Ag85B molecular mass is 3-fold higher than that of TB10.4 (Table 1). All together, spectral analyses indicate the acquisition of a well defined protein conformation for all the recombinant soluble antigenic proteins.

Bottom Line: 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).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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy. flavia.marinelli@uninsubria.it.

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.

Show MeSH
Related in: MedlinePlus