Detection of E. coli proteins

There are many examples of ELIS As used for detecting host cell impurities in the literature. Pauly et al.12 developed an ELISA to detect impurities in erythropoietin that had a detection limit of around 0.05 ng/ml. SDS polyacrylamide gel and Western blot analysis were used to confirm the spectrum of proteins detected and to demonstrate the specificity of the antibody preparation. Anicetti et al.14 describe an assay for the detection of E. coli proteins in recombinant DNA-derived human growth hormone. Whitmire and Eaton15 report on an immuno-ligand assay for quantitation of process-specific E. coli host cell contaminant proteins in a recombinant bovine somatotropin. 

Figure 3 (A) SDS PAGE of E. coli proteins 4 hours after medium shift. Lane 1 molecular weight standards 2 no isoleucine added, induction 3 isoleucine added, no induction. Lanes 4 to 9 analogs added as indicated at the bottom of the figure. (B) Autoradiogram of a western blot obtained from the gel above. DHFR was detected with antibodies raised against the amino terminal hexahistidine tag...

Direct detections of E. coli have been demonstrated since 1998, when Fratamico et al. demonstrated the detection of viable E. coli 0157 H7 using a Biacore system [18]. They compared two sensing platforms, both based on attachment via Ar-ethyl-ArC(diniethylaminopropyl)carbodiimide hydrochloride (EDC) and AT-hydroxy-succinimide (NHS) chemistry. Monoclonal or polyclonal antibody was bound directly to the sensing surface or was immo-Irilized on a bound layer of protein A or protein G. Using a sandwich assay, as seen in Fig. 1, a lower limit of detection of 5-7 x 10 cfu/mL was demonstrated. 

Following this first published study on SPR detection of E. coli 0157 Ft7, other direct detection studies have been performed. In 2002, Oh et al. used a Multiskop SPR biosensor to detect E. coli 0157 FI7 at concentrations as low as 10" cells/mL [19]. This was done using a monoclonal antibody (Mab) immobilized on a protein G surface, similar to the study by Fratamico [18]. In 2003, Oh et al. used the same sensing system as in their previous study, but altered their sensing surface to be an optimized mixed self-assembled monolayer (SAM). This lowered the detection hmit of E. coli 0157 H7 to... 

In another study (114), three different immobilization methods were evaluated for the detection of E. coli using an antibody coated crystal. The best results were obtained with a protein A coated method, and a response for 10 - lO cells 1 ml of E. coli K12 and other antigens of the Enterobacteriaceae family was observed. Repeated usage of the coated crystal by removing the bound antigen with urea or glycine HCl buffer (pH 2.6) was not very successful. 

PelZ is a hydrophilic protein of 420 amino acids with a short hydrophobic sequence at its N-terminal end which has Ae characteristics of the signal sequences of exported proteins. The signal peptide may be 24 amino acids long, which would corroborate wiA the usual length encountered in prokaryotes. The molecular cloning of the pelZ gene in an expression vector pT7-6 allowed for the specific 35S-cysteine-methionine raAo-labelling of PelZ in E. coli K38. We could detect, in crude extracts, the presence of a precursor and a mature form of PelZ. After cell fractionation, Ae mature form of PelZ could be localized in Ae periplasm of E. coli. So PelZ appears to be a protein exported by Ae Sec-dependent system of translocation. 

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