- 學位論文
Recognition and removal of bacterial endotoxin by recombinant horseshoe crab plasma lectin
利用重組鱟血漿凝集素識別及移除細菌內毒素
摘要
內毒素為革蘭氏陰性菌細胞膜之成分，會造成人體發炎反應，故為食品藥物管理局(Food and Drug Administration，FDA)法規要求必需於重組蛋白質製程中移除。內毒素具高熱穩定性，雖然可以高溫處理破壞，但同時會在生物藥生產過程中破壞蛋白質活性，因此，以專一性結合內毒素之樹脂去除內毒素為蛋白藥物製程中的重要步驟。由台灣三棘鱟(Taiwanese Tachypleus tridentatus)血漿中分離之血漿凝集素具有辨識革蘭氏陽性、陰性菌體以及其他病原菌表面之病原相關分子型態(Pathogen-associated molecular patterns，PAMPs)的能力。本實驗室成功以大腸桿菌系統表現與純化重組鱟血漿凝集素(recombinant horseshoe crab plasma lectin，rHPL)，除具有細菌脂多醣內毒素辨識活性外，亦可專一結合細菌表面之鼠李糖(rhamnose)。本研究探討rHPL對於脂多醣的最佳結合酸鹼值和溫度，且利用還原胺化反應將rHPL固定於氧化態Sepharose CL-6B樹脂製成修飾型樹脂(rHPL-CL-6B)，並驗證其成功結合綠膿桿菌之脂多醣內毒素之能力。rHPL獨特的鼠李糖及內毒素結合能力具細菌內毒素移除的潛力，此新穎內毒素識別及移除機制可貢獻於新型內毒素偵測及移除策略開發。
並列摘要
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.