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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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Time course of pH (○, solid line), dO2 (♦, solid line) and OD600nm (●, dashed line) in 3 L batch cultivation trials of recombinant E. coli BL21(DE3) cells producing M. tuberculosis immunogenic proteins. A)E. coli BL21(DE3) cells containing pET32b-Trx-TB10.4 plasmid and grown in LB medium. B)E. coli BL21(DE3) cells containing pET32b-Trx-Ag85B plasmid and grown in SB medium. C)E. coli BL21(DE3) cells containing pColdI-His-full2 and growing in SB/NaCl medium. Temperature (■, dashed line) was kept constant at 37°C before IPTG addition and then reduced to 18°C for Trx-TB10.4 and Trx-Ag85B production, and at 15°C for His-full2 production, until harvest. The induction of protein expression was done at OD600nm = 0.8 with 0.1 mM IPTG for Trx-TB10.4, at OD600nm = 2 with 0.1 mM IPTG for Trx-Ag85B, and at OD600nm = 2 with 0.1 mM IPTG for His-full2. Biomass (wet weight) collected at the harvest time was 8.5 g/L for Trx-TB10.4, 19.5 g/L for Trx-Ag85B and 6.5 g/L for His-full2.
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Figure 5: Time course of pH (○, solid line), dO2 (♦, solid line) and OD600nm (●, dashed line) in 3 L batch cultivation trials of recombinant E. coli BL21(DE3) cells producing M. tuberculosis immunogenic proteins. A)E. coli BL21(DE3) cells containing pET32b-Trx-TB10.4 plasmid and grown in LB medium. B)E. coli BL21(DE3) cells containing pET32b-Trx-Ag85B plasmid and grown in SB medium. C)E. coli BL21(DE3) cells containing pColdI-His-full2 and growing in SB/NaCl medium. Temperature (■, dashed line) was kept constant at 37°C before IPTG addition and then reduced to 18°C for Trx-TB10.4 and Trx-Ag85B production, and at 15°C for His-full2 production, until harvest. The induction of protein expression was done at OD600nm = 0.8 with 0.1 mM IPTG for Trx-TB10.4, at OD600nm = 2 with 0.1 mM IPTG for Trx-Ag85B, and at OD600nm = 2 with 0.1 mM IPTG for His-full2. Biomass (wet weight) collected at the harvest time was 8.5 g/L for Trx-TB10.4, 19.5 g/L for Trx-Ag85B and 6.5 g/L for His-full2.

Mentions: After optimization at the shaken flask-scale, processes and production media were scaled up in 3 L bioreactor. As shown by the parameters controlled on line (dO2 and pH) and by densitometric analysis of cell growth during batch cultivation (Figure 5), the growth of recombinant E. coli BL21(DE3) cells holding the pColdI-His-full2 was faster than those transformed by pET32b-Trx-TB10.4 and pET32b-Trx-Ag85B, even if a lower temperature was adopted after induction of protein expression (15°C for His-full2 production vs. 18°C for Trx-TB10.4 and Trx-Ag85B). In 6 hours from inoculum, maximum biomass was achieved and dO2 was completely depleted in cells producing His-full2. In Trx-Ag85B production, oxygen depletion occurred after 8 hours from inoculum and the phase of oxygen limitation lasted for further ten hours. E. coli Trx-TB10.4-producing cells showed a similar time course of oxygen consumption, but the levels of dO2 never decreased below 50% of saturation, indicating a minor respiratory activity; growth of the culture was slower and reached a lower level of biomass production (in wet weight: 19.5 and 8.5 g/L were recovered after 24 hours from cells producing Trx-Ag85B or Trx-TB10.4, respectively, see Table 2). For both Trx-Ag85B or Trx-TB10.4 producing cells, after an initial slight decrease, pH tended to alkalinization in the following fermentation phase. In cells producing His-full2, a marked acidification of medium and a rapid re-increase in oxygen level after the initial sharp reduction occurred, probably reflecting a stress status arresting cell proliferation after IPTG addition and growth temperature reduction. Notwithstanding the higher OD600nm of pColdI-His-full2 containing cells, final biomass was only 6.5 g/L in wet weight (Table 2).


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)

Time course of pH (○, solid line), dO2 (♦, solid line) and OD600nm (●, dashed line) in 3 L batch cultivation trials of recombinant E. coli BL21(DE3) cells producing M. tuberculosis immunogenic proteins. A)E. coli BL21(DE3) cells containing pET32b-Trx-TB10.4 plasmid and grown in LB medium. B)E. coli BL21(DE3) cells containing pET32b-Trx-Ag85B plasmid and grown in SB medium. C)E. coli BL21(DE3) cells containing pColdI-His-full2 and growing in SB/NaCl medium. Temperature (■, dashed line) was kept constant at 37°C before IPTG addition and then reduced to 18°C for Trx-TB10.4 and Trx-Ag85B production, and at 15°C for His-full2 production, until harvest. The induction of protein expression was done at OD600nm = 0.8 with 0.1 mM IPTG for Trx-TB10.4, at OD600nm = 2 with 0.1 mM IPTG for Trx-Ag85B, and at OD600nm = 2 with 0.1 mM IPTG for His-full2. Biomass (wet weight) collected at the harvest time was 8.5 g/L for Trx-TB10.4, 19.5 g/L for Trx-Ag85B and 6.5 g/L for His-full2.
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Related In: Results  -  Collection

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Figure 5: Time course of pH (○, solid line), dO2 (♦, solid line) and OD600nm (●, dashed line) in 3 L batch cultivation trials of recombinant E. coli BL21(DE3) cells producing M. tuberculosis immunogenic proteins. A)E. coli BL21(DE3) cells containing pET32b-Trx-TB10.4 plasmid and grown in LB medium. B)E. coli BL21(DE3) cells containing pET32b-Trx-Ag85B plasmid and grown in SB medium. C)E. coli BL21(DE3) cells containing pColdI-His-full2 and growing in SB/NaCl medium. Temperature (■, dashed line) was kept constant at 37°C before IPTG addition and then reduced to 18°C for Trx-TB10.4 and Trx-Ag85B production, and at 15°C for His-full2 production, until harvest. The induction of protein expression was done at OD600nm = 0.8 with 0.1 mM IPTG for Trx-TB10.4, at OD600nm = 2 with 0.1 mM IPTG for Trx-Ag85B, and at OD600nm = 2 with 0.1 mM IPTG for His-full2. Biomass (wet weight) collected at the harvest time was 8.5 g/L for Trx-TB10.4, 19.5 g/L for Trx-Ag85B and 6.5 g/L for His-full2.
Mentions: After optimization at the shaken flask-scale, processes and production media were scaled up in 3 L bioreactor. As shown by the parameters controlled on line (dO2 and pH) and by densitometric analysis of cell growth during batch cultivation (Figure 5), the growth of recombinant E. coli BL21(DE3) cells holding the pColdI-His-full2 was faster than those transformed by pET32b-Trx-TB10.4 and pET32b-Trx-Ag85B, even if a lower temperature was adopted after induction of protein expression (15°C for His-full2 production vs. 18°C for Trx-TB10.4 and Trx-Ag85B). In 6 hours from inoculum, maximum biomass was achieved and dO2 was completely depleted in cells producing His-full2. In Trx-Ag85B production, oxygen depletion occurred after 8 hours from inoculum and the phase of oxygen limitation lasted for further ten hours. E. coli Trx-TB10.4-producing cells showed a similar time course of oxygen consumption, but the levels of dO2 never decreased below 50% of saturation, indicating a minor respiratory activity; growth of the culture was slower and reached a lower level of biomass production (in wet weight: 19.5 and 8.5 g/L were recovered after 24 hours from cells producing Trx-Ag85B or Trx-TB10.4, respectively, see Table 2). For both Trx-Ag85B or Trx-TB10.4 producing cells, after an initial slight decrease, pH tended to alkalinization in the following fermentation phase. In cells producing His-full2, a marked acidification of medium and a rapid re-increase in oxygen level after the initial sharp reduction occurred, probably reflecting a stress status arresting cell proliferation after IPTG addition and growth temperature reduction. Notwithstanding the higher OD600nm of pColdI-His-full2 containing cells, final biomass was only 6.5 g/L in wet weight (Table 2).

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