Reduced toxicity of expression, in Escherichia coli, of antipollutant antibody fragments and their use as sensitive diagnostic molecules

Abstract

Single-chain antibody fragments (scAb), specific for the chlorophenoxy acid herbicide mecoprop, have been expressed and purified from the bacterium Escherichia coli. Co-expression with the colE1-compatible, arabinose-inducible, skp expression vector pHELP1 prevented bacterial lysis and significantly increased both total and functional expression yield. The periplasmic protein, SKP, may have a role as a generic detoxification protein. Surface plasmon resonance (BIAcore 2000) analysis confirmed that the purified scAb retained similar binding kinetics to the monoclonal antibody (Mab) from which it was cloned. In competition ELISA, the bacterial scAb showed the same specificity for mecoprop and a related herbicide, MCPA, as the Mab but an increase in sensitivity for free antigen in all ELISA formats. Bacterially expressed antibody fragments provide a simple, sensitive and cost-effective alternative to the traditional production of diagnostic Mabs via tissue culture.

Expression of a Functional zipFv Antibody Fragment and Its Fusions with Alkaline Phosphatase in the Cytoplasm of anEscherichia coli

“…Co-expression of chaperone proteins such as the Skp chaperone protein with antibody fragments had been reported to reduce toxicity and to enhance the proper folding of scFv or Fab fragments expressed in E. coli (35), and Levy et al demonstrated that co-expression of Skp or DsbC chaperone protein increased the cytoplasmic production of functional Fab fragments up to 5-6-fold (23). Additionally, DsbC co-expression increases the yield of tissue plasminogen activator (tPA) in the cytoplasm of E. coli by more than 20-fold (36).…”

Rapid selection of anti-hapten antibodies isolated from synthetic and semi-synthetic antibody phage display libraries expressed inEscherichia coli

“…Single E. coli XL‐1 Blue colonies containing antibodies specific for microcystin were grown overnight in 5 ml LB containing 1% (w/v) glucose, 50 μg ml −1 amp and 12.5 μg ml −1 tetracycline at 37°C using published methods[13]. Each culture was used to inoculate 50 ml Terrific broth (TB)–glu–amp–tet in 250‐ml baffled flasks and the culture grown to an OD of 15.…”

Rapid selection of anti-hapten antibodies isolated from synthetic and semi-synthetic antibody phage display libraries expressed in Escherichia coli

“…Single E. coli XL-1 Blue colonies containing antibodies speci¢c for microcystin were grown overnight in 5 ml LB containing 1% (w/v) glucose, 50 Wg ml 31 amp and 12.5 Wg ml 31 tetracycline at 37 ‡C using published methods [13]. Each culture was used to inoculate 50 ml Terri¢c broth (TB)^glu^amp^tet in 250-ml ba¥ed £asks and the culture grown to an OD of 15.…”

“…Two immunotubes were coated overnight with 100 Wg ml 31 microcystin LR^BSA in phosphate bu¡ered saline (PBS), washed with PBS and blocked with 2% skimmed milk^PBS at room temperature for 2 h. The concentrated phage particles (approximately 1U10 13 ) from each library (Gri⁄n or Tomlinson) were added to the immunotubes. Speci¢c scFv phage bound to the antigen, and the unbound phage were removed by washing.…”

“…The scAb fragments were puri¢ed via the 6-His tail using Ni 2þ charged immobilised metal a⁄nity chromatography (IMAC) Fast Flow Sepharose resin [13] and the presence of scAb detected by capture ELISA of the human CU domain [13]. The scAb concentration was calculated from a standard curve plotted using human IgG at known concentrations and corrected for di¡erences in molecular masses between IgG (150 kDa) and scAb (40 kDa).…”