An Expression System for the E-coli Fermentation of Recombinant Antibody F-ab Fragments from Mice and Rabbits

作者：Wiebe Julia C; Schueller Carolin; Reiche Jana A; Kramer Karl^*; Skerra Arne; Hock Bertold

来源：Journal of AOAC International, 2010, 93(1): 80-88.

A mutually compatible vector system was developed for the bench-top fermenter production of mouse and rabbit F-ab 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 = 20-40) of the atrazine-specific mouse F-ab fragment K411B using JM83 harboring pASK85-pro-K411B in a 2 L bench-top vessel resulted in a yield of 240 mu g/L.OD600 affinity-purified protein (13.8 mg). In contrast, expression of leporid F-ab 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 F-ab fragments together with four periplasmic folding-helper proteins encoded on pTUM4 led to a significantly improved folding efficiency, resulting in a yield of 50 mu g/L.OD600 affinity-purified rabbit F-ab fragment (3.3 mg) from the 2 L bench-top fermenter.

• 出版日期2010-2