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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-Ag85B and Ag85B. Deconvoluted spectra from intact ESI-LIT-MS analyses of: A) purified Trx-Ag85B; B) mature Ag85B after proteolytic cleavage.
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Figure 3: MS analyses of purified Trx-Ag85B and Ag85B. Deconvoluted spectra from intact ESI-LIT-MS analyses of: A) purified Trx-Ag85B; B) mature Ag85B after proteolytic cleavage.

Mentions: The synthetic cDNA coding for the mature form (285 amino acids) of Ag85B lacking the region encoding for the N-terminal 40 residues corresponding to the putative signal peptide (UniProtKB/Swiss-Prot Accession no. P0C5B9.1; PDB Accession no. 1F0N_A), also optimized for E. coli expression, was subcloned into pET32b plasmid, in frame with nucleotidic sequence coding for Trx, with a His6-tag sequence and with a sequence recognized by EK – these sequences are located at the N-terminus of Ag85B (Figure 1). The Trx-Ag85B chimeric protein is 450 amino acid long and its molecular mass is 48.4 kDa (Table 1 and Figure 1, see also Additional file 1: Figures S1 and S2). Using the same standard conditions set up for Trx-TB10.4, a very low amount of soluble Trx-Ag85B was produced and a low amount of pure protein (< 1 mg/L) was isolated by metal chelating chromatography. In order to improve the amount of protein produced, a number of expression conditions - using different growth media (LB, TB, SB and minimal medium M9), adding 0.1 or 1.0 mM IPTG when OD600nm reached 0.7, 2 or after overnight growth (saturation condition) - were tested. In all cases, following IPTG addition, cells were grown at 18°C and collected after 4 hours, and the expression level was assessed by Western blot using anti-His-tag antibody. The largest amount of soluble Trx-Ag85B was obtained growing cells in SB medium and inducing expression with 0.1 mM IPTG at an OD600nm = 2 (see Additional file 2: Figure S4). Further improvement was obtained growing cells for additional 16 hours (overnight) after IPTG addition. Using these conditions, ca. 25-30% of the chimeric protein was soluble (Additional file 2: Figure S5B and C) corresponding to a productivity of 20 mg/L: 9 mg/L of Trx-Ag85B (> 95% purity degree) were isolated after a single-step purification on nickel-affinity column chromatography (Table 2, see Additional file 2: Figure S5A, B and C). MS spectrum of the fusion protein confirmed its identity and purity (Figure 3A).


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-Ag85B and Ag85B. Deconvoluted spectra from intact ESI-LIT-MS analyses of: A) purified Trx-Ag85B; B) mature Ag85B after proteolytic cleavage.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: MS analyses of purified Trx-Ag85B and Ag85B. Deconvoluted spectra from intact ESI-LIT-MS analyses of: A) purified Trx-Ag85B; B) mature Ag85B after proteolytic cleavage.
Mentions: The synthetic cDNA coding for the mature form (285 amino acids) of Ag85B lacking the region encoding for the N-terminal 40 residues corresponding to the putative signal peptide (UniProtKB/Swiss-Prot Accession no. P0C5B9.1; PDB Accession no. 1F0N_A), also optimized for E. coli expression, was subcloned into pET32b plasmid, in frame with nucleotidic sequence coding for Trx, with a His6-tag sequence and with a sequence recognized by EK – these sequences are located at the N-terminus of Ag85B (Figure 1). The Trx-Ag85B chimeric protein is 450 amino acid long and its molecular mass is 48.4 kDa (Table 1 and Figure 1, see also Additional file 1: Figures S1 and S2). Using the same standard conditions set up for Trx-TB10.4, a very low amount of soluble Trx-Ag85B was produced and a low amount of pure protein (< 1 mg/L) was isolated by metal chelating chromatography. In order to improve the amount of protein produced, a number of expression conditions - using different growth media (LB, TB, SB and minimal medium M9), adding 0.1 or 1.0 mM IPTG when OD600nm reached 0.7, 2 or after overnight growth (saturation condition) - were tested. In all cases, following IPTG addition, cells were grown at 18°C and collected after 4 hours, and the expression level was assessed by Western blot using anti-His-tag antibody. The largest amount of soluble Trx-Ag85B was obtained growing cells in SB medium and inducing expression with 0.1 mM IPTG at an OD600nm = 2 (see Additional file 2: Figure S4). Further improvement was obtained growing cells for additional 16 hours (overnight) after IPTG addition. Using these conditions, ca. 25-30% of the chimeric protein was soluble (Additional file 2: Figure S5B and C) corresponding to a productivity of 20 mg/L: 9 mg/L of Trx-Ag85B (> 95% purity degree) were isolated after a single-step purification on nickel-affinity column chromatography (Table 2, see Additional file 2: Figure S5A, B and C). MS spectrum of the fusion protein confirmed its identity and purity (Figure 3A).

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