Elimination of trace endotoxin protein from rough chemotype LPS

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Abstract
The phenol-chloroform-petroleum ether (PCP) extraction is the method of choice for isolating LPS chemotypes from rough mutant bacteria. However, we have observed that a high percentage of PCP-purified LPS preparations contain trace levels of protein contaminants, and these protein contaminants may exhibit endotoxin-like activity. To obtain protein-free rough LPS, a modified phenol-water extraction procedure was developed for use as a final step to follow PCP extraction. Using this procedure, Salmonella minnesota Ra, Rc, Rd and Re LPS were recovered in yields of 82-100% from the original PCP-extracted preparations. Yields were determined by recovery of KDO and supported by analysis of silver stained SDS-PAGE gels. Although the original PCP-purified chemotypes were contaminated by major 41, 38.5, 29.5, 23, 17 and 14 kDa proteins (detected by Western blotting and gold staining), the repurified LPS contained no detectable protein. The repurified LPS retained all of its original bioactivity as defined by the ability to stimulate normal C3H/OuJ macrophages to secrete TNF. The importance of utilizing repurified LPS in biologic studies was illustrated by the loss of > 99% of activity when repurified LPS was used to stimulate TNF secretion by LPS 'hyporesponsive' C3H/HeJ macrophages.

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