that can express disulfide-bonded proteins as intracellular, soluble, properly-folded proteins. Unlike comparable strains, we achieve high cell densities, making it commercially viable for recombinant protein expression. Furthermore, the bacteria have been engineered to efficiently cleave the N-terminal methionine found on cytoplasmic E. coli proteins. To create the strain, we deleted the gor gene in BL21(DE3), resulting in an oxidative cytoplasm. The deleted gor gene was replaced with a methionine peptidase with the same promoter as found on the plasmid containing the recombinant protein gene so that peptidase is co-expressed when needed. We call the strain Gor/Met for the deleted and replaced genes. Remarkably, Gor/Met can grow to &gt;300g/L in a fed-batch fermentation. We have initially used the Gor/Met strain to address the need for low-cost “carrier proteins”, a critical requirement for affordable conjugate vaccines. Conjugate vaccines consist of carbohydrates, peptides, or other poorly immunogenic antigens chemically linked to a carrier protein, which provides T cell help. Conjugate vaccines have proven remarkably effective in preventing disease but are costly to develop and manufacture. Fina Biosolutions focuses on the research and development of conjugate vaccines. Among the proteins expressed are CRM197 (EcoCRM®) and recombinant tetanus toxin heavy chain C (rTTHc) and a genetically detoxified tetanus toxin, M8TT. EcoCRM® is produced at ~ 2 g/L fermentation culture in the Gor/Met strain. It has been extensively compared to CRM197 obtained from multiple manufacturers (Hickey et al., J Pharm Sci. 107, 1806, 2018) and 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. rTTHc is a 52 kDa protein produced at &gt;0.5 g/L. 8MTT is a 150 kDa protein with 5 disulfide bond pairs, also purified at &gt;0.5g/L fermentation. All three proteins were found by mass spec analysis to have extremely low levels of N-terminal methionine, showing that amino acid was efficiently cleaved by the peptidase. 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.

Engineered E. coli with an oxidative cytoplasm and efficient cleavage of N-terminal methionine

ACS Fall 2021

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