AIMS:
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.
METHODS AND RESULTS:
The use of co-expression of a sulfhydryl oxidase and protein disulfide isomerase for the production of soluble CRM197 in E. coli is described. CRM197 contains two disulfide 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... More
AIMS:
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.
METHODS AND RESULTS:
The use of co-expression of a sulfhydryl oxidase and protein disulfide isomerase for the production of soluble CRM197 in E. coli is described. CRM197 contains two disulfide 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 CRM197 compared to CRM197 commercially produced in cultures of recombinant Pseudomonas fluorescens indicated that the E. coli soluble protein compares favorably on all structural levels.
CONCLUSIONS:
Sulfhydryl oxidase and protein disulfide isomerase are enzymes involved in the formation of intra-protein disulfide 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 CRM197 in E. coli, which was previously unachievable.
SIGNIFICANCE AND IMPACT OF STUDY:
Previous literature reports have shown that CRM197 can be expressed in Escherichia coli, though only in an insoluble form, or in soluble form as a fusion protein. It is currently commercially produced in cultures of recombinant Pseudomonas 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 .
