Improved production of single domain antibodies with two disulfide bonds by co-expression of chaperone proteins in the Escherichia coli periplasm

• Thermal stabilized single domain antibodies (sdAb) are valuable immunoreagents.
• Mutagenesis to insert an extra disulfide bond, DSB, is known to stabilize sdAb.
• Stability due to the additional DSB results in deceased yields due incorrect DSBs.
• Co-expression of four chaperones using helper plasmid pTUM4 improves yields.
• pTUM4 improves the yield of sdAb possessing both natural and created extra DSB.

Single domain antibodies are recombinantly expressed variable domains derived from camelid heavy chain antibodies. Natural single domain antibodies can have noncanonical disulfide bonds between their complementarity-determining regions that help position the binding site. In addition, engineering a second disulfide bond serves to tie together β-sheets thereby inhibiting unfolding. Unfortunately, the additional disulfide bond often significantly decreases yield, presumably due to formation of incorrect disulfide bonds during the folding process. Here, we demonstrate that inclusion of the helper plasmid pTUM4, which results in the expression of four chaperones, DsbA, DsbC, FkpA, and SurA, increased yield on average 3.5-fold for the nine multi-disulfide bond single domain antibodies evaluated. No increase in production was observed for single domain antibodies containing only the canonical disulfide bond.