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Efficient bioprocess for high-density fermentation and rapid purification of soluble tag-free CRM197 protein expressed in Escherichia coli
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.
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