479696 Optimizing Expression of Therapeutic Antigen-Binding Fragments (Fab) in E. coli

Monday, November 14, 2016
Grand Ballroom B (Hilton San Francisco Union Square)
Jeremy Adams, School for Engineering of Matter, Transport, and Energy, Arizona State University, Tempe, AZ and Brent L. Nannenga, Chemical Engineering, Arizona State University, Tempe, AZ

Antigen-binding fragments (Fab), proteins containing the functional region of antibodies, have been proposed as potential therapeutics for neurodegenerative diseases such as Alzheimer’s. However, overexpression of Fabs has proven to be difficult. Previously, it has been demonstrated that certain folding engineering strategies have significantly improved functional expression of secreted proteins. This project tests the effect that the secretion pathway and the presence of trigger factor have on the overall expression of Fabs. Genes encoding the light and heavy chains of the C6T Fab were cloned into the pBLN-200 vector, each preceded by the same signal sequence, using Gibson Assembly. This was done with both the stII signal sequence (directing through the Sec-dependent pathway) and the DsbA signal sequence (directing through the SRP-dependent pathway). Both new plasmids were transformed into two different E. coli strains: BW25113, containing trigger factor, labeled tig+, and KTD101, a BW25113 derivative lacking trigger factor, labeled Δtig. These four systems were expressed at 37ºC, and cell growth was closely monitored. Interestingly, the Δtig-DsbA system, which was expected to have the highest efficiency of protein expression, produced the poorest cell growth, while the traditional tig+-stII system demonstrated the most robust cell growth following induction. Future work will examine the levels of functional Fab overexpression and Fab export to the periplasm, which will help to explain the reason for this decrease in cell growth.

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