Purification and Characterization of Shigalike toxins from food borne E coli isolates

Abstract

Out of a total of 100 pani samples collected from different street vendors of newlinepanipuri in Bangalore as part of earlier research work 74 isolates of E. coli were newlineobtained. Five E. coli isolates pe16, pe68, pe74, pe88 and pe93 that showed multidrug newlineresistance, higher concentraction of protein and positive for haemolysis were selected newlinefor the present investigation. On genomic DNA isolation and electrophoresis all the five newlineE. coli isolates pe16, pe68, pe74, pe88 and pe93 showed a single prominent band on newlineagarose gel. On PCR amplification of stx1 gene for the isolates there was no amplified newlineproduct for stx1 in any of the samples. Only primer dimers were observed in all the newlinesamples. However, there was an amplified product of amplicon size between 100 bp to newline200 bp corresponding for stx2f from the E. coli isolate pe16, a band of amplicon size newlinebetween 600 bp to 700 bp for hlyA from the E. coli isolate pe68, two bands with the newlineamplicon sizes between to 100 bp to 200 bp for stx2f and stx2c respectively from the E. newlinecoli isolate pe74, a band with the amplicon size between 100 bp to 200 bp for stx2c newlinefrom the E. coli isolate pe88 and two bands with the amplicon sizes between 100 bp to newline200 bp for stx2f and stx2 from the E. coli isolate pe93. When the proteins from the newlineisolates were subjected to partial purification by ammonium sulphate precipitation newlinefollowed by dialysis, multiple protein bands were observed. Crude samples from all the newlinefive isolates showed more bands compared to ammonium sulphate precipitated and newlinedialysed samples. The protein fractions collected after purification by ion-exchange newlinechromatography were subjected to protein estimation. Fractions that showed highest newlineprotein concentration were selected for further studies. The fraction number 8 from newlineanion exchange chromatography with 1.38 mg/ml of protein from the isolate pe16, the newlinefraction number 6 from anion exchange chromatography with 2.33 mg/ml of protein newlinefrom the isolate pe68, the fraction number 7 from cation exchange chromatography with newline1.54 mg/ml of protein from the isolate pe74 the fraction number 5 from cation exchange newlinechromatography with 3.21 mg/ml of protein from the isolate pe88 and the fraction newlinenumber 7 from anion exchange chromatography with 3.55 mg/ml of protein from the newlineisolate pe93 were used for further studies. The protein fractions from the above step of newlinepurification were subjected to SDS-PAGE along with protein marker. Staining with newlineCoomassie blue after electrophoresis showed prominent bands.