Abstract
Endotoxins are cell membrane components of Gram negative bacteria, which often induce human inflammatory responses. Therefore, endotoxin removal is demanded by Food and Drug Administration (FDA) during production of recombinant proteins. Since endotoxins are highly thermostable, high temperature is required to destroy them. However, high temperature also abolishes protein activity during production of gene-based products such as biologics. Accordingly, resin-based endotoxin removal strategy is commonly used to pharmaceutical proteins at laboratory scale. Recombinant horseshoe crab plasma lectin (rHPL) derived from hemolymph of Taiwanese Tachypleus tridentatus recognizes pathogen-associated molecular patterns (PAMPs) including LPSs of Gram negative bacteria as well as lipoteichoic acids (LTAs) of Gram-positive bacteria. rHPL has been successfully expressed and purified from Escherichia coli system, and it binds to L-rhamnose-containing component on bacterial surface. In this study optimal pH and temperature for LPS binding activity of rHPL are investigated and rHPL has been immobilized onto oxidized Sepharose CL-6B resin by reductive amination, leading to generation of rHPL-CL-6B resin for successful capture of LPSs of 2 Pseudomonas aeruginosa strains. rHPL possesses unique surface L-rhamnose and endotoxin binding activities. Such specific characteristics make rHPL potential for detection and removal of bacteria endotoxin. Our novel endotoxin recognition and removal mechanisms by rHPL can significantly contribute to development of novel endotoxin detection and removal strategies.