Protein and endotoxin interactions and endotoxin removal from protein solutions
Department of Chemical Engineering, Chemistry and Environmental Science
Doctor of Philosophy
Luo, Robert G.
Kebbekus, Barbara B.
Meegoda, Jay N.
Sirkar, Kamalesh K.
Trattner, Richard B.
The investigation objectives of this study include: endotoxin disaggregation by proteins; protein concentration effect on protein-endotoxin binding and endotoxin removal; size exclusion high performance liquid chromatography (SE-HPLC) studies on endotoxin dissociation by bovin serum albumin (BSA); use of Ca+2 to re-aggregate endotoxin in hemoglobin solutions and the subsequent removal of endotoxin by ultrafiltration; quantitative determination of Ca+2 effects on endotoxin removal and protein yield in a two-stage ultrafiltration.
It is known that some proteins can disaggregate endotoxins and form complexes with lipopolysaccharides (LPS). It is also known that Ca can act as "bridges" to aggregate LPS in aqueous solutions. However many questions remain unanswered. What are the effects of protein concentration on such LPS disaggregation? Can Ca act as "bridges" to re-aggregate LPS subunits in protein solution where LPS vesicles have been disaggregated by protein molecules into small fragments? How will the presence of Ca+2 affect the endotoxin removal and protein recovery? This study will investigate these problems.
The effects of protein concentration on endotoxin disaggregation, protein-LPS binding, and endotoxin removal have been investigated. It was found that hemoglobin A0 and albumin could disaggregate LPS and their concentrations had significant effects on protein-LPS interaction. In contrast, lysozyme and cytochrome C did not disaggregate LPS.
In the study of size exclusion high performance liquid chromatography (SEHPLC) studies on endotoxin dissociation by bovine serum albumin (BSA), it was found that BSA could disaggregate LPS. BSA concentration had a significant effect on the LPS disaggregation. Pre-incubation of the BSA-LPS mixture could enhance the LPS disaggregation.
Ca+2 was used to re-aggregate endotoxin in hemoglobin solutions and to subsequently remove endotoxin by ultrafiltration. It was found that Ca could reaggregate LPS in protein solutions where LPS had been disaggregated by hemoglobin A0. Such re-aggregation improved the endotoxin removal efficiency by ultrafiltration.
In the study of quantitative determination of Ca+2 effects on endotoxin removal and protein yield in a two-stage ultrafiltration, it was found Ca+2 would re-aggregate LPS from protein solution. In the presence of Ca+2, endotoxin removal efficiency was increased without sacrificing the protein yield.
njit-etd1999-035 (150 pages ~ 6,715 KB pdf)
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Created July 27, 2004