Soluble Expression of CRM197 with Periplasmic Expression Option in E.coli

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Soluble Expression of CRM197 with Periplasmic Expression Option in E.coli

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Background:

CRM197, which retains the same inflammatory and immune-stimulant properties as diphtheria toxin but with reduced toxicity, has been used as a safe carrier in conjugated vaccines. Recently, CRM197 was produced as a single peptide where fragment A was linked to fragment B, forming an intramolecular disulphide bond. In addition to its vaccine adjuvant properties, there is also growing interest in CRM197 because of its potential antitumor activity related to its ability to bind the soluble form of HB-EGF, which is highly expressed in some human cancers.

Rationale:

Soluble CRM197 production has been expressed in multiple hosts such as Bacillus subtilis, P. fluorescens, Pichia pastoris, and C. diphtheriae. However, these attempts were partially unsuccessful or did not generate yields sufficient for industrial-scale production. Attempts conducted in Escherichia coli led to limited protein expression due to the formation of inclusion bodies. To overcome this, expression was also attempted in Bacillus subtilis using the subtilisin signal sequence for secretion into the culture medium. However, the maximum yield was about 7.1 mg/L. Secretion can decrease the costs of protein recovery. Likewise, in E. coli, the secretion of CRM197 protein to the periplasmic space has been considered a possible approach to decreasing protein recovery costs.

Approach:

Premas Biotech has optimized the parameters for the expression and purification of soluble recombinant CRM197. The synthetic CRM197 gene was cloned into pET series vector for periplasmic expression by using appropriate restriction endonucleases. The resulting recombinant plasmid was transformed into BL21ʎDE3 strain of E. coli followed by a selection of transformants on selective antibiotic marker agar plates and expression was studied using scouting parameters applied for the best clone selection.

Fully soluble recombinant CRM197 was obtained in high yields and with an authentic N‐terminus by targeting the protein to the periplasm of E. coli using the signal recognition particle dependent signal sequence. Expression of soluble CRM197 was confirmed by immunoblotting in periplasmic fractions (Figure 1 and 2). Single peaks by SEC-HPLC and RP-HPLC confirmed the purity of the protein (Figure 2). Various feeding strategies and additives in the growth and induction phase, with different media combinations and maintenance of process parameters like pH, DO, Air to achieve, temperature allowed the process development of r-CRM197 at larger scale in the fermenter. The process developed by Premas is robust and linearly scalable, which will reduce costs of production of future vaccines using CRM17 as a carrier in the polysaccharide-protein conjugate.

Fig 1: SDS PAGE (upper panel) and Immunoblot analysis (Lower Panel) for rCRM197

Periplasmic Extraction

Reducing Non-reducing

Measurable Impact:

1) Expression of soluble and highly purified CRM197
2) scalable for preclinical and clinical studies
3) Cost-effective process which is the essential requirement for commercial purposes
4) Will reduce cost of future vaccines which are using CRM17 as a carrier in polysaccharide-protein conjugate.

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