Extraction and purification of lipopolysaccharide from Escherichia coli (local Isolate) and study its pyrogenic activity

Abstract

In this study, we tried to extract and purify the LPS from E. coli local isolate and determine the molecular weight, purity, and pyrogenic effect of the product and compare it with standard E. coli O55:B5 LPS, the E. coli LPS was extracted by using hot phenol method then SDS- PAGE was used with both Coomassie blue and silver nitrate stain to determine its molecular weight and protein contamination also we used HPLC to the estimation of E. coli LPS purity and finally the pyrogenicity of extracted E. coli LPS was tested by using rabbit pyrogen test. The result showed that the hot phenol method with enzymatic treatment gave highly pure LPS with a high yield reach up to 242.4 mg, staining the SDS page gel with Coomassie blue and silver nitrate uncover the high purification of the extracted LPS (ELPS) with no protein contamination, with a molecular weight range between 15-23 kDa, HPLC test reveals that purity of ELPS was 100 % compared with standard LPS. The rabbits' pyrogen test confirmed that the biological activity of ELPS. In conclusion, the LPS was extracted with high purity compare with standard LPS and without any protein or DNA contamination by using the hot phenol method also the extracted rough LPS was slightly lighter than the standard LPS used but this did not affect its biological activity which remained intact

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