Removal of lipopolysaccharides from protein-lipopolysaccharide complexes by nonflammable solvents.
During the recovery of recombinant proteins from gram negative bacteria, many of the methods used to extract proteins from cells release lipopolysaccharides (LPS, endotoxin) along with the protein of interest. In many instances, LPS will co-purify with the target protein due to specific or non-specific protein-LPS interactions. We have investigated the ability of alkanediols to effect the separation of LPS from protein-LPS complexes while the complexes are immobilized on ion exchange chromatographic resins. Proteins were complexed with fluorescently labeled LPS and bound to ion exchange resin. Alkanediol washes of the resins were preformed and the proteins eluted. Column eluates were monitored for LPS and protein by fluorescence and UV spectroscopy, respectively. Alkanediols were effective agents for dissociating LPS from protein-LPS complexes. The efficiency of LPS removal increased with increasing alkanediol chain length. The 1,2-alkanediol isomers were more effective than terminal alkanediol isomers in the separation of LPS from protein-LPS complexes, while the separation of LPS from protein-LPS complexes was more efficient on cation exchangers than on anion exchangers. In addition, it was noted during these investigations that the 1,2-alkanediols increased the retention time of the proteins on the ion exchange resins. Alkanediols provide a safer alternative to the use of other organics such as alcohols or acetonitrile for the separation of LPS from protein due to their lower toxicity and decreased inflammability. In addition, they are less costly than many of the detergents that have been used for similar purposes.
Lin
Miao-Fang
MF
Purification Process Development Department Diosynth Biotechnology, 3000 Weston Parkway, Cory, NC 27513, USA.
Williams
Christie
C
Murray
Michael V
MV
Ropp
Philip A
PA
D001896:Boron Compounds / Q000737:chemistry; D002852:Chromatography, Ion Exchange / Q000379:methods*; D003902:Detergents; D006018:Glycols; D006586:Hexanes; D008070:Lipopolysaccharides / Q000302:isolation & purification*; D010420:Pentanes; D011994:Recombinant Proteins / Q000302:isolation & purification*; D012997:Solvents
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D003902:Detergents; D006018:Glycols; D006586:Hexanes; D008070:Lipopolysaccharides; D010420:Pentanes; D011994:Recombinant Proteins; D012997:Solvents; C578469:1,5-pentanediol; C119102:1,2-hexanediol