Variable domains of camelid heavy chain antibodies (VHHs) are the smallest available antigen binding fragments. The GRAS organism Saccharomyces cerevisiae is often used for the production of VHHs. The production yield in this organism often is too low for commercially viable large scale application as, for example, a therapeutic agent. We compare two VHHs, one of which can be produced up to 150 mg/l while the other is produced in amounts that are hardly detectable, to shed light on molecular and biological origins of those VHH production yield differences. Sequence characteristics, structure properties, and growth conditions were tested. We found that codon optimization is much more important for the gene encoding the badly produced VHH then the good produced VHHs. Even more important is to avoid VHHs in with the FW4 is encoded by germline J segment 7 which sequence contains K and L on positions 120 and 123 respectively. Moreover we validated earlier findings that ethanol improves the yield of VHHs expressed in S. cerevisiae, even for VHHs that contain only one S-S bridge. Electron microscopy showed that bad yields of secreted VHHs coincide with increased intracellular levels of (most likely) aggregated VHHs; however the level of intracellular VHHs is heterogeneously distributed of the cells of the same population
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