June 12 – 17, 2022
We have developed a novel E. coli expression system, which we call the Gor/Met strain. Gor/Met E. coli has an oxidative cytoplasm and can express disulfide-bonded proteins as intracellular, soluble, properly-folded proteins. In addition to the oxidative cytoplasm, the strain has been engineered to efficiently cleave the N-terminal methionine found on cytoplasmic E. coli proteins. To create an oxidative cytoplasm, we deleted the gor gene in BL21 E. coli, resulting in an oxidative cytoplasm. We used the gor- strain to produce the conjugate vaccine carrier protein CRM197 in a fermenter. CRM197 was expressed as a soluble, properly folded protein in the cytoplasm and no inclusion bodies were formed. We developed a simple purification process that resulted in 2 g of purified protein per liter fermentation broth. FinaBio’s CRM197 (marketed as EcoCRM®) has been extensively compared to CRM197 obtained from multiple manufacturers (Hickey et al., J Pharm Sci. 107, 1806, 2018). It was found to have the correct disulfide bonding. EcoCRM® is being used for several conjugate vaccines in development targeting S. pneumoniae, Group B Strep, malaria and vaccines for drugs of abuse. Clinical grade EcoCRM® is available now. To further improve the strain, the deleted gor gene was replaced with a methionine peptidase with the same tac promoter as the plasmid containing the recombinant protein gene. Induction induced co-expression of the methionine amino peptidase along with the heterologous recombinant protein on the plasmid. Gor/Met E. coli achieves can grow to high cell densities in a fermenter, unlike comparable strains, making it commercially viable for recombinant protein expression. Remarkably, Gor/Met can grow to >300g/L in fed-batch fermentation. We have initially used the Gor/Met strain to produce a unique, genetically detoxified tetanus toxin (8MTT, Przedpelski et al. mBio Aug 11;11(4):e01668-20). 8MTT is purified at >0.5 g/L fermentation and was found by mass spec analysis to have extremely low levels of N-terminal methionine, showing that the amino acid was efficiently cleaved by the peptidase. 8MTT was found to be comparable to tetanus toxoid as a carrier protein. Gor/Met E. coli was used to express CRM197, tetanus toxin heavy chain fragment C and human IL-10. This work shows the potential of the Gor/Met E. coli to be a commercially viable production strain for producing high yields of disulfide-bonded proteins, with their native sequence. Among these are carrier proteins such as CRM197 and a new genetically detoxified tetanus toxin, along with many therapeutic proteins of interest. The availability of affordable conjugate vaccine carrier proteins produced in a low-cost expression system makes the development of conjugate vaccines more feasible. The variety of applications including vaccines for malaria, S. pneumonia, Covid and other infectious diseases as well as vaccines for areas as diverse as drugs of abuse, cancer and cholesterol control.
Andrew Lees, Morgane Ollivault-Shiflett, Min-Ju Chang, and Renaud Jacquemart, "Genetically detoxified diphtheria and tetanus toxins, EcoCRM197® and 8MTT, expressed in an engineered E. coli strain and use as conjugate vaccine carrier proteins" in "Vaccine Technology VIII", Tarit Mukhopadhyay, Merck Research Laboratories, USA; Charles Lutsch, Sanofi Pasteur, France; Linda Hwee-Lin Lua, University of Queensland, Australia; Francesc Godia, Universitat Autònoma de Barcelona, Spain Eds, ECI Symposium Series, (2022). https://dc.engconfintl.org/vaccine_viii/48