AnalyticalRemoval of Endotoxin from Recombinant Protein Preparations
Introduction
Endotoxin is a lipopolysaccharide (LPS) present in the cell wall of most gram-negative bacteria including Escherichia coli. It has long been known that when gram-negative bacteria gain access to the blood stream of the mammalian host, inflammation and septic shock may occur [1]. These effects were caused by endotoxins that elicited the production and systemic release of inflammatory mediators by LPS responsive cells of the mammalian host [2]. In addition, nanogram quantities of endotoxin can affect the biochemical events of certain cells and cell-free systems [3], and can interfere with in vitro experiments. Endotoxin can readily contaminate water, aqueous solutions, and buffers. Recombinant proteins derived from E. coli are often contaminated with endotoxins since the cell wall is known to contain large quantities of lipopolysaccharides [4]. The presence of even small amounts of endotoxin from preparations of recombinant proteins can cause adverse reactions such as shock when these proteins are used as antigens for immunizing experimental animals 4, 5. Due to those adverse reactions, it is essential to remove endotoxins from drugs, injectabables, and other biological and pharmaceutical products. Some commonly used techniques for removing endotoxin contaminants are ultrafiltration [6]and ion exchange chromatography [7]. In the case of macromolecules such as protein, ultrafiltration would not be applicable while ion exchange is a less specific method. The use of polymyxin B and histidine affinity chromatography, and Triton X-114 phase separation have been more successful in removing endotoxins from protein preparations 8, 9, 10. This article evaluates these three procedures for removing endotoxin contaminants from large scale preparation of recombinant proteins purified from E. coli, in searching an effective way to reduce the endotoxin to a level that experimental animals can tolerate.
Section snippets
Chemicals and Supplies
Pyrotell™ LAL for gel-clot assay, and LAL reagent water (LRW) were purchased from Associates of Cape Cod (Woods Hole, MA, USA). Flint glass reaction tubes and Eppendorf tubes were purchased from Fisher Scientific (Toronto, ON). Sterile pipette tips were purchased from VWR (Toronto, ON). Sterile cryovials were purchased from Simport Plastics (Beloeil, Que). Histidine-sepharose, polymyxin b-sepharose, Triton X-114, and deoxycholate were purchased from Sigma Chemicals (St. Louis, MO, USA).
Reagent Preparation
LAL Gel-Clot Assay and Endotoxin Levels in Protein Buffers
The LAL gel-clot assay determines endotoxin levels by the ability of minute amounts of lipopolysaccharides to induce a gel-clot when in contact with Limulus blood. The clotting reaction is catalyzed by a cascade of enzyme activations occurring in the granules of amebocytes. The amount of endotoxin present in the test solution is directly correlated to the gel formation process.
Using the LAL assay, we found detectable levels of endotoxin in a number of buffers used to store the recombinant
Discussion
We have demonstrated that by performing three cycles of Triton X-114 phase separation, endotoxin levels in recombinant proteins derived from E. coli were reduced by as much as 99%. Further reduction can be obtained if more cycles of phase separation were performed, as shown with rcTnI. Both immunoactivity and biological activity of recombinant cTnI subjected with Triton X-114 phase separation were not affected by this process. Removal of endotoxin from rcTnI solution by affinity chromatography
Acknowledgements
We thank Margaret Dodge, Joan Mangion, and Jianying Yang for demonstrating repeatedly that Triton X-114 phase separation effectively removes endotoxin from other recombinant protein preparations and for their valuable discussions. The authors also wish to thank Julie Wiard for the CK-MB Stratus® assay.
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