Lipopolysaccharide removal affinity matrices based on novel cationic amphiphilic peptides.

Lipopolysaccharide (LPS) is one of the most challenging contaminants in biopharmaceutical industries. Cationic amphiphilic peptides (CAPs) -based affinity matrices can be potent tools for LPS removal in such situations. In this study, two newly designed CAPs derived from the LPS binding site of factor C of horseshoe crab S3E3 and S3E3A were immobilized chemo-selectively on diaminodipropylamine (DADPA) and iodoacetyl functionalized Sepharose beads. Both peptides were immobilized via their carboxyl or sulfhydryl (thiol) groups by amide or thioether bonds, respectively. The generated four affinity matrices were used to remove LPS from bovine serum albumin (BSA). The effects of different influential factors including pH, NaCl, Ethylenediaminetetraacetic acid (EDTA), and LPS concentrations on LPS removal efficiency and protein recovery were investigated by Plackett Burman (PB) method.Statistical analysis revealed that immobilized S3E3 removed LPS more effectively than immobilized S3E3A. Increasing pH and LPS concentration had negative effects on LPS removal efficiency and protein recovery. Increasing NaCl concentration improved protein recovery but reduced LPS removal efficiency. Other factors such as EDTA and type of buffer had no significant effect on the measured responses.

Effect of glutamic acid elimination/substitution on the biological activities of S3 cationic amphiphilic peptides.

Cationic amphiphilic peptides (CAPs) are usually classified as bacterial membrane targeting molecules. Rational design and modification of cationic and amphiphilic properties of CAPs have made them to be used in new medical and biotechnological applications. However, CAPs modification and development strategies are challenging issues due to the risk of cytotoxicity or hemolytic activity. In this research, modified variants of S3 peptide were introduced. S3 is a linear 34 amino acid peptide derived from the lipopolysaccharide (LPS) binding site of factor C in horseshoe crab's hemolymph. Net positive charges of variants (S3E3 and S3E3A) increased by either eliminating negatively charged residues of the peptides or substituting them with alanine. Different biological activities of new variants including LPS binding affinity, antimicrobial activity, cytotoxicity against human breast tumor cell line, and hemolytic property were studied and compared to those of S3 peptide. S3E3 variant showed 68.5% higher LPS binding affinity, 40.4% stronger anti-microbial activity, conserved hemolytic property with the same anti-cancer activity compared to S3peptide. These results revealed that elimination/substitution of negatively charged residues will be a proper strategy for modification of S3 peptide.