Overexpression and purification of the recombinant diphtheria toxin variant CRM197 in Escherichia coli

Stefan, Alessandra; Conti, Matteo; Rubboli, Diego; Ravagli, L; Presta, Enrica; Hochkoeppler, Alejandro

doi:10.1016/j.jbiotec.2011.08.024

Cited by 35 publications

(34 citation statements)

References 24 publications

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“…Previous reports on the expression of CRM 197 in E. coli (Stefan et al . ), indicated the native nontagged version could not be successfully expressed, and CRM 197 fused to a histidine affinity tag could be expressed only in an insoluble form in BL21(DE3). Mahamad et al .…”

Section: Discussionmentioning

confidence: 99%

“…Previous reports on the expression of CRM 197 in E. coli (Stefan et al 2011), indicated the native nontagged version could not be successfully expressed, and CRM 197 fused to a histidine affinity tag could be expressed only in an insoluble form in BL21(DE3). Mahamad et al (2016) also reported production of a fusion thioredoxin-His-CRM 197 protein in the trxB and gor mutant E. coli Origami B(DE3), when co-expressed with different chaperones in a E. coli host strain.…”

Section: Discussionmentioning

confidence: 99%

“…Presently produced commercially by isolation from cultures of recombinant Pseudomonas fluorescens, reports in the literature show that CRM 197 has successfully been expressed in Escherichia coli, although in an insoluble form, and the presence of a six-histidine affinity tag was required for this successful expression (Stefan et al 2011). Escherichia coli is a widely used system for heterologous expression, but it has its drawbacks, particularly for those proteins which contain disulphide bonds (Stewart et al 1998), which are crucial for proper folding.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Co-expression of sulphydryl oxidase and protein disulphide isomerase inEscherichia coliallows for production of soluble CRM₁₉₇

et al. 2017

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Previous literature reports have shown that CRM can be expressed in E. coli, though only in an insoluble form, or in soluble form as a fusion protein. It is currently commercially produced in cultures of recombinant P. fluorescens. The use of a widely used, well-characterized expression host such as E. coli, rather than P. fluorescens broadens the applicability of the production technology, and the production system described here is worthy of further investigation for scaled up manufacture of CRM .

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Co-expression of sulphydryl oxidase and protein disulphide isomerase inEscherichia coliallows for production of soluble CRM₁₉₇

et al. 2017

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“…Typical limitations of E. coli include protein mis-folding and aggregation, proteolysis, and the lack of disulfide bond formation [21]. CRM197 is a challenging protein to produce in E. coli: the protein contains two disulfide bonds -which cannot be formed efficiently in the reducing cytoplasmic environment -and is prone to mis-folding, resulting in total or partial insolubility [9,10]. In extreme cases, CRM197 is not produced at all, despite codon optimization [9].…”

Section: Advantages Of Periplasmic Vs Cytoplasmic Crm197 Productionmentioning

confidence: 99%

High‐yield production of recombinant CRM197, a non‐toxic mutant of diphtheria toxin, in the periplasm of Escherichia coli

Goffin

Dewerchin

Rop

et al. 2017

Biotechnology Journal

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A high cell density fed-batch process was developed for production of recombinant CRM197, a non-toxic mutant of diphtheria toxin widely used as a carrier in polysaccharide-protein conjugate vaccines. Fully soluble recombinant CRM197 was obtained in high yields and with an authentic N-terminus, by targeting the protein to the periplasm of Escherichia coli using the Signal Recognition Particle (SRP)-dependent signal sequence of FlgI. Response Surface Methodology (RSM) was used to optimize the set-points of key process parameters (pH and feed rate at induction). Optimal production of periplasmic CRM197 was found at a slightly basic pH (7.5). The feed rate during induction was positively correlated with the accumulation of unprocessed cytoplasmic CRM197, consistent with limited capacity of the SRP secretion pathway. Decreasing the feed rate to align the protein synthesis rate with the secretion capacity, resulted in minimal production of cytoplasmic CRM197. Besides, the host background was found critical for production of periplasmic CRM197: B834(DE3) was the highest producer (>3 g/L), while BLR(DE3) produced one third less CRM197, and very low yields (290 mg/L) were obtained with HMS174(DE3). The optimized process is robust and linearly scalable, and represents a 20-fold yield improvement compared to a process based on Corynebacterium diphtheriae.

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“…However, the recombinant protein was insoluble and always found inside protein aggregates. The expression of CRM197 devoid of the short tag always failed [Stefan et al, 2011]. Pfenex Inc. has developed a Pseudomonas fluorescens strain that generates high levels of CRM197.…”

Section: Production Of Cross-reacting Materials 197mentioning

confidence: 99%

Recent Approaches in Vaccine Development against <b><i>Streptococcus pneumoniae</i></b>

Tarahomjoo

2014

Microb Physiol

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Streptococcuspneumoniae is a major cause of morbidity and mortality among children under 5 years of age worldwide. Vaccines have long been used for protection against pneumococcal infections. Capsular polysaccharides of pneumococci are main antigenic components of these vaccines. However, pneumococcal polysaccharide-based vaccines are not able to elicit appropriate immunological responses in young children and cannot induce the immune memory. Thus, pneumococcal conjugate vaccines were developed through chemical coupling of an immunogenic carrier protein to the capsule. The currently available pneumococcal conjugate vaccines elicited protection against the bacterium efficiently. However, these vaccines are expensive to manufacture and have limited serotype coverage. In this mini-review, therefore, we describe approaches attempted by researchers to circumvent the shortcomings of the conjugate vaccines including specifying appropriate cultivation conditions for the production of S. pneumoniae capsular antigens, development of suitable expression systems for the frequently used carrier protein in the conjugate vaccines (cross-reacting material 197), construction of protein-based vaccines, whole-cell vaccines, DNA vaccines, and using antigen delivery vehicles. Future trends in this field are also discussed.

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Overexpression and purification of the recombinant diphtheria toxin variant CRM197 in Escherichia coli

Cited by 35 publications

References 24 publications

Co-expression of sulphydryl oxidase and protein disulphide isomerase inEscherichia coliallows for production of soluble CRM₁₉₇

Co-expression of sulphydryl oxidase and protein disulphide isomerase inEscherichia coliallows for production of soluble CRM₁₉₇

High‐yield production of recombinant CRM197, a non‐toxic mutant of diphtheria toxin, in the periplasm of Escherichia coli

Recent Approaches in Vaccine Development against <b><i>Streptococcus pneumoniae</i></b>

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Overexpression and purification of the recombinant diphtheria toxin variant CRM197 in Escherichia coli

Cited by 35 publications

References 24 publications

Co-expression of sulphydryl oxidase and protein disulphide isomerase inEscherichia coliallows for production of soluble CRM197

Co-expression of sulphydryl oxidase and protein disulphide isomerase inEscherichia coliallows for production of soluble CRM197

High‐yield production of recombinant CRM197, a non‐toxic mutant of diphtheria toxin, in the periplasm of Escherichia coli

Recent Approaches in Vaccine Development against <b><i>Streptococcus pneumoniae</i></b>

Contact Info

Product

Resources

About

Co-expression of sulphydryl oxidase and protein disulphide isomerase inEscherichia coliallows for production of soluble CRM₁₉₇

Co-expression of sulphydryl oxidase and protein disulphide isomerase inEscherichia coliallows for production of soluble CRM₁₉₇