A mutually compatible vector system was developed for the bench-top fermenter production of mouse and rabbit Fab fragments comprising pASK85-pro-K411B and pASK85Rab-pro-WR13. These vectors provide a mouse- and rabbit-specific cloning site, respectively, the tetA promoter/operator and the proBA operon that complements the Pro biosynthetic deficiency of the Escherichia coli strain JM83 and can serve as an additional selection marker. Fermentation at elevated cell density (OD600 = 2040) of the atrazine-specific mouse Fab fragment K411B using JM83 harboring pASK85-pro-K411B in a 2 L bench-top vessel resulted in a yield of 240 g/L OD600 affinity-purified protein (13.8 mg). In contrast, expression of leporid Fab frag... More
A mutually compatible vector system was developed for the bench-top fermenter production of mouse and rabbit Fab fragments comprising pASK85-pro-K411B and pASK85Rab-pro-WR13. These vectors provide a mouse- and rabbit-specific cloning site, respectively, the tetA promoter/operator and the proBA operon that complements the Pro biosynthetic deficiency of the Escherichia coli strain JM83 and can serve as an additional selection marker. Fermentation at elevated cell density (OD600 = 2040) of the atrazine-specific mouse Fab fragment K411B using JM83 harboring pASK85-pro-K411B in a 2 L bench-top vessel resulted in a yield of 240 g/L OD600 affinity-purified protein (13.8 mg). In contrast, expression of leporid Fab fragments using pASK85Rab-pro-WR13 was unsuccessful due to the aggregation of rabbit light chains, which probably relates to a general problem of this specific class of immunoglobulins with their additional Cys residues. Coexpression of rabbit Fab fragments together with four periplasmic folding-helper proteins encoded on pTUM4 led to a significantly improved folding efficiency, resulting in a yield of 50 g/L OD600 affinity-purified rabbit Fab fragment (3.3 mg) from the 2 L bench-top fermenter.
