To make protein samples suitable for animal studies and cell culture, contaminat-ing pyrogens, such as lipopolysaccharide (endotoxin) from gram negative bacte-ria, must be removed. Here we describe the use and optimization of a peptide with high affinity and specificity for endotoxin in column chromatography to pro-duce endotoxin-free samples with high protein recovery.
Removal of endotoxin from solutions for animal studies, cell culture, transplanta-tion, stem cell technologies, cell sorting, and other mammalian cell treatments is a priority. The majority of lipid in the outer membrane of gram negative bacteria consists of lipopolysaccharide (LPS), also called endotoxin. Sub-nanogram lev-els of endotoxin can trigger immune responses and alter the phenotype and func-tion of many cells including monocytes, neutrophils, dendritic cells, hepatocytes, vascular and respiratory epithelium, and arterial smooth muscle cells. For years, the Limulus amoebocyte lysate (LAL) test has been the standard for detecting even trace amounts of endotoxin. This test was developed from observations that horseshoe crab amoebocytes aggregate and degranulate in response to LPS as a defense mechanism against gram-negative bacteria [1,2]. This degranulation re-leases a series of enzymes that include Factor C, the initial activator of a serine protease cascade [3,4]. In the LAL assay, Factor C detects picogram levels of LPS and initiates a clotting reaction. In recent years, the assay has been modified for detection with fluorescence, colorimetry, and turbidity, which made it more quantitative and less open to interpretation. Recently, a 34 amino acid LPS-binding Sushi domain was identified in Factor C. Expression and characteriza-tion of this linear peptide showed high binding to (Kd 10-6-10-8) and neutralization of (ENC50 2.25 μM) LPS . BioDtech’s EndoBind-R™ is a DADPA-agarose-conjugated S1 peptide affinity chromatography column. It has been used to re-move endotoxin from water, buffers, and cell culture media. Under optimized conditions, it may also be used to produce endotoxin-free protein solutions with high recovery. Additionally, the S1 peptide is highly resistant to a wide range of pH’s and ionic strengths making it suitable for many applications in which tradi-tional chromatography methods, such as ion exchangers, affinity ultrafiltration, and immunoaffinity matrices are not applicable [6,7].