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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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MS analyses of purified Trx-TB10.4 and TB10.4. Deconvoluted spectra from intact ESI-LIT-MS analyses of: A) Trx-TB10.4; B) purified TB10.4 before the proteolytic cleavage optimization; C) purified TB10.4 after the proteolytic cleavage optimization and D) the same sample after further incubation at 37°C, for 24 hours.
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Figure 2: MS analyses of purified Trx-TB10.4 and TB10.4. Deconvoluted spectra from intact ESI-LIT-MS analyses of: A) Trx-TB10.4; B) purified TB10.4 before the proteolytic cleavage optimization; C) purified TB10.4 after the proteolytic cleavage optimization and D) the same sample after further incubation at 37°C, for 24 hours.

Mentions: The synthetic cDNA coding for TB10.4 (GenBank Accession no. CAA17363.1) was optimized to match the codon usage for E. coli expression (see Additional file 1: Figures S1 and S2). Codon usage optimization was essential since E. coli is a low G + C content (~ 50%) Gram-negative bacterium while M. tuberculosis is a high G + C (> 65%) Gram-positive actinobacterium. The cDNA was subcloned into pET32b plasmid in frame with nucleotidic sequence coding for thioredoxin (Trx), with a His6-tag sequence and with a sequence recognized by enterokinase (EK) – these sequences are located at the N-terminus of TB10.4 (Figure 1). Trx is a 12 kDa protein that remarkably increases the solubility of fusion proteins [13]. The chimeric Trx-TB10.4 protein (261 amino acids, molecular mass 28.1 kDa, Table 1 and Figure 1) was produced in E. coli using BL21(DE3) strains. Basal expression was performed in LB medium, adding 0.1 mM IPTG when OD600nm reached 0.6-0.8 and collecting cells after additional 4 hours of growth at 18°C: using these conditions, a fairly good amount of chimeric protein was produced as soluble form and about 2 mg/L of > 85% pure Trx-TB10.4 was purified after a single-step purification on nickel-affinity chelating column. Optimization of the expression conditions (i.e. growing cells for 16 hours at 18°C after IPTG addition) increased significantly the production of the soluble form of the chimeric protein: 12 mg/L of > 95% pure Trx-TB10.4 was purified by metal-chelating chromatography (Table 2, see Additional file 2: Figures S3A, B and C). Mass spectrometry (MS) spectrum of the fusion protein reported in Figure 2A confirmed the identity and purity of the fused protein, indicating a molecular mass corresponding to the isoform lacking methionine at the N-terminal.


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)

MS analyses of purified Trx-TB10.4 and TB10.4. Deconvoluted spectra from intact ESI-LIT-MS analyses of: A) Trx-TB10.4; B) purified TB10.4 before the proteolytic cleavage optimization; C) purified TB10.4 after the proteolytic cleavage optimization and D) the same sample after further incubation at 37°C, for 24 hours.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: MS analyses of purified Trx-TB10.4 and TB10.4. Deconvoluted spectra from intact ESI-LIT-MS analyses of: A) Trx-TB10.4; B) purified TB10.4 before the proteolytic cleavage optimization; C) purified TB10.4 after the proteolytic cleavage optimization and D) the same sample after further incubation at 37°C, for 24 hours.
Mentions: The synthetic cDNA coding for TB10.4 (GenBank Accession no. CAA17363.1) was optimized to match the codon usage for E. coli expression (see Additional file 1: Figures S1 and S2). Codon usage optimization was essential since E. coli is a low G + C content (~ 50%) Gram-negative bacterium while M. tuberculosis is a high G + C (> 65%) Gram-positive actinobacterium. The cDNA was subcloned into pET32b plasmid in frame with nucleotidic sequence coding for thioredoxin (Trx), with a His6-tag sequence and with a sequence recognized by enterokinase (EK) – these sequences are located at the N-terminus of TB10.4 (Figure 1). Trx is a 12 kDa protein that remarkably increases the solubility of fusion proteins [13]. The chimeric Trx-TB10.4 protein (261 amino acids, molecular mass 28.1 kDa, Table 1 and Figure 1) was produced in E. coli using BL21(DE3) strains. Basal expression was performed in LB medium, adding 0.1 mM IPTG when OD600nm reached 0.6-0.8 and collecting cells after additional 4 hours of growth at 18°C: using these conditions, a fairly good amount of chimeric protein was produced as soluble form and about 2 mg/L of > 85% pure Trx-TB10.4 was purified after a single-step purification on nickel-affinity chelating column. Optimization of the expression conditions (i.e. growing cells for 16 hours at 18°C after IPTG addition) increased significantly the production of the soluble form of the chimeric protein: 12 mg/L of > 95% pure Trx-TB10.4 was purified by metal-chelating chromatography (Table 2, see Additional file 2: Figures S3A, B and C). Mass spectrometry (MS) spectrum of the fusion protein reported in Figure 2A confirmed the identity and purity of the fused protein, indicating a molecular mass corresponding to the isoform lacking methionine at the N-terminal.

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