@ARTICLE{Singh2026-bx,
  title     = "Efficient bioprocess for high-density fermentation and rapid
               purification of soluble tag-free {CRM197} protein expressed in
               Escherichia coli",
  author    = "Singh, Shubham and Mehta, Jaideep and Mishra, Ravi P N",
  abstract  = "Cross-reacting material 197, or CRM197, is a genetically
               detoxified variant of diphtheria toxin that is an integral part
               of several approved polysaccharide-conjugate vaccines against
               encapsulated pathogenic bacteria. CRM197 is a high-value protein
               needed as a carrier protein in enormous quantities for the
               manufacturing of conjugate vaccines, which are critical
               components of global immunisation programs, with billions of
               doses required each year. However, the scarcity of high-yielding
               clones, along with the inherent difficulties of soluble protein
               production and purification, necessitates the intercession of an
               efficient bioprocess to meet this gap. In the current
               investigation, we designed a codon-optimized gene construct
               (named pET30a\_crm197) and generated a clone in the E.
               coli-SHuffle T7 strain that expressed a very high quantity of
               tag-free soluble recombinant CRM197 protein. With the use of
               this E. coli clone, a robust inventive fermentation process was
               designed and carried out, producing a remarkable yield of
               soluble recombinant CRM197 (up to about 1500 mg/L). Controlled
               fermentation conditions and rational tweaking to the culture
               media's carbon, nitrogen, and trace elements were part of the
               process of optimizing protein expression. Subsequently, an
               interpreted, rapid, and high-efficiency protein purification
               process was developed, which uses tangential-flow filtration
               (TFF) followed by single-step ion-exchange chromatography to
               produce bioactive and appropriately folded CRM197 protein of
               high purity. The physicochemical and functional properties of
               purified CRM197 were determined using various analytical
               techniques, which confirmed the protein's identity, purity,
               stability, bioactivity, and secondary structure. This overall
               work offers vaccine developers a cost-effective, scalable, and
               integrated bioprocess for high-yield soluble production and
               purification of rCRM197 carrier protein.",
  journal   = "Int. J. Biol. Macromol.",
  publisher = "Elsevier BV",
  volume    =  338,
  number    = "Pt 2",
  pages     = "149685",
  month     =  jan,
  year      =  2026,
  keywords  = "Bioactive; High-purity; High-yield; One-step purification;
               SHuffle T7 strain; Soluble expression",
  language  = "en"
}