title: Improvements in the cell-free production of functional antibodies using cell extract from protease-deficient Escherichia coli mutant.
abstract: Expression of a functional antibody fragment (Fab) using an Escherichia coli cell-free expression system has been reported previously [Jiang et al., FEBS Lett., 514, 290-294 (2002)]. The low yield of the synthesized antibody, however, limits the usefulness of the cell-free expression system, partly due to the degradation of product by endogenous proteases from the E. coli extract. To determine which proteases are responsible for the degradation, we compared the expression of a 6D9 Fab fragment under conditions whereby several protease inhibitors were added into the cell-free system. The addition of serine protease inhibitor increased the amount of the Fab fragment, indicating that serine proteases caused the antibody degradation. Therefore, several serine protease-deficient mutants of E. coli BW25113 were constructed by targeted homologous recombination. The use of extract from a double protease-deficient mutant (DeltadegP-ompT) significantly increased the amount and antigen-binding activities of an anti-HSA scFv and a 6D9 Fab fragment. These results suggest that the DegP- and OmpT-deleted mutant is a useful source of S30 extract for the production or screening of antibodies using the cell-free expression system.
authors.affiliation: Laboratory of Molecular Biotechnology, Graduate School of Biological and Agricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
authors.forename: Muhamad
authors.initials: M
authors.lastname: Ali
authors.forename: Hirotatsu
authors.initials: H
authors.lastname: Suzuki
authors.forename: Takako
authors.initials: T
authors.lastname: Fukuba
authors.forename: Xiuping
authors.initials: X
authors.lastname: Jiang
authors.forename: Hideo
authors.initials: H
authors.lastname: Nakano
authors.forename: Tsuneo
authors.initials: T
authors.lastname: Yamane
mesh_terms: D000917:Antibody Formation / Q000502:physiology*; D002474:Cell-Free System / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics; D009154:Mutation; D010447:Peptide Hydrolases / Q000172:deficiency* / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D010447:Peptide Hydrolases