Optimizing expression of Nanobody® molecules in Pichia pastoris through co-expression of auxiliary proteins under methanol and methanol-free conditions
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (Arcadia Science)
Abstract
Background
Ablynx NV, a subsidiary of Sanofi, has a long-standing focus on the development of Nanobody® molecules as biopharmaceuticals (Nanobody® is a registered trademark of Ablynx NV). Nanobody molecules are single variable domains, and they have been met with great success part due to their favorable expression properties in several microbial systems. Nevertheless, the search for the host of the future is an ongoing and challenging process. Komagataella phaffi ( Pichia pastoris ) is one of the most suitable organisms to produce Nanobody molecules. In addition, genetic engineering of Pichia is easy and an effective approach to improve titers.
Results
Here we report that P. pastoris engineered to co-express genes encoding four auxiliary proteins (HAC1, KAR2, PDI and RPP0), leads to a marked improvement in the expression of Nanobody molecules using the AOX1 methanol induction system. Titer improvement is mainly attributed to HAC1, and its beneficial effect was also observed in a methanol-free expression system.
Conclusion
Our findings are based on over a thousand fed-batch fermentations and offer a valuable guide to produce Nanobody molecules in P. pastoris . The presented differences in expressability between types of Nanobody molecules will be helpful for researchers to select both the type of Nanobody molecule and Pichia strain and may stimulate further the development of a more ecological methanol-free expression platform.
Article activity feed
-
-
Thank you for this great work! I enjoyed reading your article and I'm happy there are people working on developing and improving protein expression systems. If I understand correctly from your methods, the culture volumes you used for protein expression are large (250ml, 2L or 5L). Have you tried your expression optimization approach in a 96-well plate or a 96-deep-well block? I understand this would affect the final protein titers but it may be great for quick screening of conditions. Thank you for your time!
-
Thank you for this great work! I enjoyed reading your article and I'm happy there are people working on developing and improving protein expression systems. If I understand correctly from your methods, the culture volumes you used for protein expression are large (250ml, 2L or 5L). Have you tried your expression optimization approach in a 96-well plate or a 96-deep-well block? I understand this would affect the final protein titers but it may be great for quick screening of conditions. Thank you for your time!
-
