Most lipid in the outer membrane of gram-negative bacteria consists of lipopolysaccharide (LPS), also known as endotoxin. Endotoxin can trigger immune responses in mammals, causing pyrogenic and shock reactions. Removal of endotoxin from solutions used in animal studies, cell culture, transplantation, stem cell research, cell sorting, and other mammalian cell treatments is a priority. General methods for endotoxin removal include ultrafiltration, solvent extraction, heat sterilization, solid phase adsorption, size exclusion, ion exchange, hydrophobic interaction, and reversed phase chromatography. Selective adsorption has been shown to be the most effective technique to remove endotoxin from final solutions of biological products. Chromatography-based endotoxin removal techniques with strong selectivity can achieve low residual endotoxin levels without affecting protein recovery.
The Limulus amoebocyte lysate (LAL) test has been the standard for detecting even trace amounts of endotoxin. Its molecular mechanism of coagulation involves a protease cascade, and this cascade is based on three serine protease zymogens including factor C, factor B, proclotting enzyme, and one clottable protein, coagulogen. As the initial activator of the clotting cascade, factor C functions as a biosensor that responds to endotoxins. The endotoxin binding property of factor C resides in the amino-terminal region spanning 333 amino acids. This short region constitutes a signal peptide, a cysteine-rich region, followed by epidermal growth factor-like domain, and finally three Sushi domains. A 34-amino acid Sushi domain with high affinity for endotoxin was identified. Expression and characterization of this linear peptide showed high binding to (Kd 10-6-10-8) and neutralization of (ENC50 2.25 μM) endotoxin. It has been used to remove endotoxin from water, buffer, and cell culture media, and also to remove endotoxin from protein and DNA solutions with minimal product loss.
Amerigo Scientific offers EndoBind-R™, a DADPA agarose-conjugated Sushi peptide (S1 peptide) affinity chromatography column. The initial binding between the S1 peptide and endotoxin is due to electrostatic interactions between the positively charged residues near the N-terminus of the peptide and the negatively charged phosphoryl head groups of the endotoxin. Then, hydrophobic interactions between the C-terminal end of S1 peptide and the acyl chains of endotoxin strengthen the binding. To optimize recovery conditions, the buffer pH needs to be adjusted near the isoelectric point (pI) of the protein being purified to minimize electrostatic interactions. And, the salt concentration required for optimal protein recovery should be established. Since the S1 peptide is not affected by a wide array of buffering parameters, it is easy to establish protein-specific conditions that provide high protein recovery and endotoxin removal with endotoxin levels below 0.01 EU/ml.
Figure 1. EndoBind-R™
Figure 2. Steps to Optimize Conditions
EndoBind-R™ are DADPA-agarose conjugated S1 peptide affinity chromatography columns bound to a 4% cross-linked beaded agarose support resin. They can be used under a broad range of conditions with high specificity, without specific equipment or buffer requirements. Removal of endotoxin using EndoBind-R™ is fast, easy, and inexpensive. Single-Use EndoBind-R™ has a lower capacity to bind endotoxin than standard EndoBind-R™ and cannot be reused or regenerated.
|EndoBind-R™ (1 ml column)||1 ml pre-packed column|
|EndoBind-R™ (5 ml column)||5 ml pre-packed column|
|EndoBind-R™ Single-Use (1 ml column)||1 ml pre-packed column|
|EndoBind-R™ Single-Use (6 x 1 ml columns)||6 x 1 ml pre-packed column|
EndoBind-R™ can be used to remove endotoxin from a broad range of aqueous solutions including buffers, cell culture media, protein samples, DNA preparations and therapeutic compounds. Under optimized conditions, EndoBind-R™ may also be used to produce endotoxin-free protein solutions with high recovery. The S1 peptide is highly resistant to a wide range of pH and ionic strengths, making it suitable for many applications in which traditional chromatography methods such as ion exchangers, affinity ultrafiltration, and immunoaffinity matrices are not suitable.
Protein purification with EndoBind-R™ resulted in high protein recovery of samples and removal of endotoxin to sub-picogram levels. Efficient endotoxin removal owes to the high affinity of the S1 peptide on the column to the lipid A portion of endotoxins. This binding occurs via initial electrostatic interactions, which is reinforced by subsequent hydrophobic interactions. Because of the nature of this binding scheme, the pI of the protein being purified and the salt and pH conditions of the buffer are critical.
In the examples given, after optimal purification conditions were determined, peak fraction protein recovery was usually in excess of 70% and total protein recovery approached 100% in all but one example (Table 1). In all protein solutions tested, endotoxin was removed to below the detection limit of 0.01 EU/ml (1 picogram/ml). This demonstrated that EndoBind-R™ could remove endotoxin efficiently in a wide range of buffers, pH values, and salt concentrations. These results included loads in excess of 750 EU/ml which contain endotoxin from two distinct sources. Similar purification was achieved using samples with endotoxin levels ranging from 10 to 10,000 EU/ml and protein concentrations as high as 40 mg/ml.
Table 1. Summary of Protein Recovery and Endotoxin Removal with EndoBind-R™
|Protein||Recovery (%)||Initial Endotoxin (EU/ml)||Endotoxin After EndoBind-R™ (EU/ml)|
The methods used here are also applicable to "ramp-up" experiments for larger scale preparations. In addition to the parameters addressed here, other variables such as chelating agents, detergents, specific buffer and salt ions, and protein concentrations can be adjusted to meet specific protein criteria.
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