S7: Binary expression phenomena for high titer and scalable production of a fusion-free cysteine knot protein in E. coli

Cysteine knots are structurally complex proteins resulting from triple beta-sheet structures held together by three intramolecular disulfide bonds yielding an inherently stable fold. The standard for recombinant microbial synthesis of cysteine knots is the expression of the peptide of interest via fusion to a carrier protein that directs the fusion to form inclusion bodies (IBs). Due to high costs of enzymatic cleavage, expression with a fusion-tag is not economically viable for large scale production. Here, the synthesis of an already challenging cysteine knot, further complicated by its homodimerized structure containing 7 disulfide bonds, is presented. Initial attempts to produce the knot with a standard IB process were unsuccessful. A first round of plasmid screening showed that a single base pair (bp) change in the ribosome binding site (RBS) and codons following ATG led to binary changes in expression from “off” to “on”. Produced IBs, were insufficient in size for large scale disc-stack harvest. A second round of cloning further changing the upstream sequence showed that few bp changes to reduce the hairpin loop conformation played a crucial role in expression enhancement. Ultimately, a bicistronic vector where a small peptide was coded upstream of the cysteine knot yielded a strain with the highest expression levels. Cell line screening efforts were conducted in the Micro-24 high-throughput system. Bench-top 1L reactors were used for optimization of fermentation conditions which further enhanced expression. Production of the cysteine knot in E. coli was increased from none to 2 g/L and scaled successfully for program supply.