High‐level accumulation of a recombinant antibody fragment in the periplasm of <i>Escherichia coli</i> requires a triple‐mutant (<i>degP prc spr</i>) host strain

<jats:title>Abstract</jats:title><jats:p>During production of a humanized antibody fragment secreted into the periplasm of <jats:italic>Escherichia coli</jats:italic>, proteolytic degradation of the light chain was observed. In order to determine which protease(s) were responsible for this degradation, we compared expression of the F(ab′)<jats:sub>2</jats:sub> antibody fragment in several <jats:italic>E. coli</jats:italic> strains carrying mutations in genes encoding periplasmic proteases. Analysis of strains cultured in high cell density fermentations showed that the combination of mutations in <jats:italic>degP prc spr</jats:italic> was necessary for the cells to produce high levels of the desired recombinant antibody fragment. In order to eliminate the possible effects of mutations in other genes, we constructed <jats:italic>E. coli</jats:italic> strains with protease mutations in isogenic backgrounds and repeated the studies in high cell density fermentations. Extensive light chain proteolysis persisted in <jats:italic>degP</jats:italic> strains. However, light chain proteolysis was substantially decreased in <jats:italic>prc</jats:italic> and <jats:italic>prc spr</jats:italic> strains, and was further decreased with the introduction of a <jats:italic>degP</jats:italic> mutation in <jats:italic>prc</jats:italic> and <jats:italic>prc spr</jats:italic> mutant strains. These results show that the periplasmic protease Prc (Tsp) is primarily responsible for proteolytic degradation of the light chain during expression of a recombinant antibody fragment in <jats:italic>E. coli</jats:italic>, and that DegP (HtrA) makes a minor contribution to this degradation as well. The results also show that <jats:italic>spr</jats:italic>, a suppressor of growth defects in <jats:italic>prc</jats:italic> strains, is required for a <jats:italic>prc</jats:italic> mutant to survive throughout high cell density fermentations. © 2004 Wiley Periodicals, Inc.</jats:p>