title: Co-expression of sulphydryl oxidase and protein disulphide isomerase in Escherichia coli allows for production of soluble CRM197.
abstract: OBJECTIVE
  To investigate the production of soluble cross-reacting material 197 (CRM197) in Escherichia coli, a safe and effective T-cell-dependent protein carrier for polysaccharides used in the manufacture and application of multivalent conjugate vaccines.
  
  
  RESULTS
  The use of co-expression of a sulphydryl oxidase (SOX) and protein disulphide isomerase for the production of soluble CRM197in E. coli is described. CRM197contains two disulphide bonds, which are normally unable to form in the reducing environment of the E. coli cytoplasm. It was found that co-expression yielded soluble CRM197, at a production rate ~10% of the production of insoluble CRM197, in equivalent small-scale cultures. Structural analysis of the purified CRM197compared to CRM197commercially produced in cultures of recombinant Pseudomonas fluorescens indicated that the E. coli soluble protein compares favourably on all structural levels.
  
  
  CONCLUSIONS
  SOX and protein disulphide isomerase are enzymes involved in the formation of intra-protein disulphide bonds, and can influence the tertiary structure of the protein being produced, resulting in increased solubility due to the correct folding of the protein. Their use enabled the production of soluble untagged CRM197in E. coli, which was previously unachievable.
  
  
  CONCLUSIONS
  Previous literature reports have shown that CRM197can 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 CRM197.
authors.affiliation: Biosciences, Council for Scientific and Industrial Research, Pretoria, South Africa.
authors.forename: R
authors.identifier: http://orcid.org/0000-0002-6486-7549
authors.initials: R
authors.lastname: Roth
authors.affiliation: Biosciences, Council for Scientific and Industrial Research, Pretoria, South Africa.
authors.forename: P
authors.initials: P
authors.lastname: van Zyl
authors.affiliation: Biosciences, Council for Scientific and Industrial Research, Pretoria, South Africa.
authors.forename: T
authors.initials: T
authors.lastname: Tsekoa
authors.affiliation: Biosciences, Council for Scientific and Industrial Research, Pretoria, South Africa.
authors.forename: S
authors.initials: S
authors.lastname: Stoychev
authors.affiliation: Biosciences, Council for Scientific and Industrial Research, Pretoria, South Africa.
authors.forename: S
authors.initials: S
authors.lastname: Mamputha
authors.affiliation: Biosciences, Council for Scientific and Industrial Research, Pretoria, South Africa.
authors.forename: S
authors.initials: S
authors.lastname: Buthelezi
authors.affiliation: Biosciences, Council for Scientific and Industrial Research, Pretoria, South Africa.
authors.forename: M
authors.initials: M
authors.lastname: Crampton
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D019704:Protein Disulfide-Isomerases / Q000235:genetics* / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D050862:Oxidoreductases Acting on Sulfur Group Donors
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D010088:Oxidoreductases; D019704:Protein Disulfide-Isomerases; D011994:Recombinant Proteins; D050862:Oxidoreductases Acting on Sulfur Group Donors; C028581:CRM197 (non-toxic variant of diphtheria toxin)
keywords: E. coli; cross-reacting material 197; disulphide bond formation; protein disulphide isomerase; soluble production; structural integrity; sulphydryl oxidase