Heterologous production and secretion of Clostridium perfringens β-toxoid in closely related Gram-positive hosts

Nijland, Reindert; Lindner, Cordula; Hartskamp, Mariska van; Hamoen, Leendert W.; Kuipers, Oscar P.

doi:10.1016/j.jbiotec.2006.07.014

Cited by 38 publications

(26 citation statements)

References 43 publications

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“…Furthermore, htrA and htrB were upregulated, as was cssR, part of the two-component system known to regulate htrA and htrB expression (7). An upregulation of htrA upon expression of ␤-toxoid, as monitored by a PhtrA-lacZ fusion, was previously described (21). Clearly, this upregulation is lower for the wild-type ␤-toxin, since a difference in htrA expression is found in the array comparison.…”

Section: Resultsmentioning

confidence: 59%

“…Recently, we have described the difficulties in secretion of Clostridium perfringens ␤-toxoid by Bacillus subtilis (21). Yet, it has been reported that wild-type ␤-toxin protein can be efficiently expressed in B. subtilis (26).…”

Section: Resultsmentioning

confidence: 99%

“…Point mutations have been introduced that render this toxin no longer toxic but still immunogenic. However, this altered ␤-toxoid is very poorly secreted by B. subtilis (21).…”

mentioning

confidence: 98%

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Changing a Single Amino Acid in Clostridium perfringens β-Toxin Affects the Efficiency of Heterologous Secretion by Bacillus subtilis

Nijland

Heerlien

Hamoen

et al. 2007

Appl Environ Microbiol

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Achieving efficient heterologous protein production and secretion by Bacillus subtilis is an attractive prospect, although often disappointingly low yields are reached. The expression of detoxified Clostridium perfringens ␤-toxin (␤-toxoid) is exemplary for this. Although ␤-toxin can be efficiently expressed and secreted by Bacillus subtilis, the genetically detoxified, and industrially interesting, ␤-toxoid variant is difficult to obtain in high amounts. To optimize the expression of this putative vaccine component, we studied the differences in the global gene regulation responses of B. subtilis to overproduction of either ␤-toxin or ␤-toxoid by transcriptomics. A clear difference was the upregulation of the CssRS regulon, known to be induced upon secretion stress, when ␤-toxoid is produced. YkoJ, a protein of unknown function, was also upregulated, and we show that its expression is dependent on cssS. We then focused on the heterologous protein itself and found that the major secretion bottleneck can be traced back to a single amino acid substitution between the ␤-toxin and the ␤-toxoid, which results in the rapid degradation of ␤-toxoid following secretion across the cytoplasmic membrane. In contrast to ␤-toxin, ␤-toxoid protein is more prone to degradation directly after secretion, most likely due to poor folding characteristics introduced with point mutations. Our results show that although the host can be adapted in many ways, the intrinsic properties of a heterologous protein can play a decisive role when optimizing heterologous protein production.

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Section: Resultsmentioning

confidence: 59%

Section: Resultsmentioning

confidence: 99%

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Changing a Single Amino Acid in Clostridium perfringens β-Toxin Affects the Efficiency of Heterologous Secretion by Bacillus subtilis

Nijland

Heerlien

Hamoen

et al. 2007

Appl Environ Microbiol

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“…After extraction of pJETAα recombinant cloning vector, Nucleotide sequence and a 364-amino acid protein sequence was deposited into the GenBank. In silico analysis of these sequences showed several putative conserved domains (46).…”

Section: Molecular Biology Studies On Clostridium To Produce Vaccine mentioning

confidence: 99%

Isolation, Specification, Molecular Biology Assessment and Vaccine Development of Clostridium in Iran: A Review

Langroudi

2015

Int J Enteric Pathog

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Context: The genus Clostridium, which consists of spore-forming anaerobes, can cause different diseases in domestic animals and human and some of them are serious and fatal. According to the increasing economic value of the meat and milk-producing animals, the importance of a certain number of such diseases in Iran is unquestionable. Evidence Acquisition: In Iran, and probably in other Near East countries, much attention was formerly paid to control more serious contagious diseases, such as rinderpest, anthrax, etc. resulting in the negligence of diseases such as enterotoxaemia. The epizootiological position has now changed whereby some of the contagious diseases are eradicated or are being methodically controlled. Now it is time to care about the other problems such as clostridial diseases, which threaten the health of the sheep and cattle. It is impossible to eradicate these infectious microorganisms, since they are normally found in the soil and the intestinal contents of apparently healthy animals. Therefore, it is necessary to resort to vaccination which in some cases has given encouraging results. To avoid the losses from such infections it is necessary to have the best possible vaccination information, methodically and regularity of the susceptible animals. Conclusions: This review refers to the veterinary aspects of the anaerobic clostridial diseases and vaccine development concerning the works carried out in Iran and especially at the Razi Serum and Vaccine Research Institute in the last eight decades.

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“…toxins is laborious and poorly predictable. [10] We have previously constructed a recombinant Escherichia coli, designated as B1-EP that expresses C. perfringens type B b1Àe toxin. [11] It was constructed by transformation of the plasmid pET-32a with the genes encoding C. perfringens type B b1Àe protein.…”

Section: Introductionmentioning

confidence: 99%

Optimization of culture conditions to improve the expression level of beta1–epsilon toxin ofClostridium perfringenstype B inEscherichia coli

Chao

Cheng

Wang

et al. 2016

Biotechnology & Biotechnological Equipment

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The detoxified beta1Àepsilon (b1Àe) toxin protein of Clostridium perfringens type B provides protection from C. perfringens types B, C and D infections. Acetate is the primary by-product from the cell growth and expression of b1Àe protein. In the present study, the effects of pH and dissolved oxygen (DO) on the expression of b1-e protein were investigated. Two-stage pH and DO control strategies were developed for the expression of b1Àe protein. The obtained results indicated that higher cell density and concentration of b1-e protein, and lower accumulation of acetate were obtained when pH was maintained at a constant level of 6.5 (0À6 h) and 7.0 (6À16 h), and the DO level was maintained at 60% (0À6 h) and 30% (6À16 h). Furthermore, the impact of intermittent, DO feedback, pH feedback and glucose-stat feeding on the expression of b1Àe protein were studied. By using the DO feedback feeding, combined with the stage control of pH (6.5 for 0À6 h, 7.0 for 6À16 h) and DO (60% for 0À6 h, 30% for 6À16 h), the highest cell density of 2.045 (absorbance at 600 nm) and a b1Àe protein concentration of 63.24 mg/L were obtained, and the accumulation of acetate decreased to 0.872 g/L.

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Heterologous production and secretion of Clostridium perfringens β-toxoid in closely related Gram-positive hosts

Cited by 38 publications

References 43 publications

Changing a Single Amino Acid in Clostridium perfringens β-Toxin Affects the Efficiency of Heterologous Secretion by Bacillus subtilis

Changing a Single Amino Acid in Clostridium perfringens β-Toxin Affects the Efficiency of Heterologous Secretion by Bacillus subtilis

Isolation, Specification, Molecular Biology Assessment and Vaccine Development of Clostridium in Iran: A Review

Optimization of culture conditions to improve the expression level of beta1–epsilon toxin ofClostridium perfringenstype B inEscherichia coli

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