Removal of endotoxin from recombinant protein preparations.
OBJECTIVE
To develop an effective method to remove endotoxin from large scale E. coli recombinant protein purifications.


METHODS
Triton X-114 phase separation, affinity chromatography utilizing immobilized polymyxin B or immobilized histidine, were used to remove endotoxin from purified preparations of recombinant CK-BB, CK-MB, CK-MM, myoglobin, and cardiac troponin I. Endotoxin levels were measured by a Limulus Amebocyte Lysate gel-clot assay. The immunoactivity of these protein preparations was determined by BIAcore analysis using a panel of in-house generated monoclonal antibodies and by a Stratus Fluorometric Analyzer. In the case of troponin I, the BIAcore was also utilized to measure troponin C interactions.


RESULTS
Phase separation with Triton X-114 was the most effective method in reducing the amount of endotoxin present in the protein preparations compared to either polymyxin B or histidine affinity chromatography. With Triton X-114, the reduction in endotoxin levels was greater than 99% and recovery of the proteins after endotoxin removal was greater than 90%. All three procedures for removing endotoxin had no deleterious effects on the immunoactivity of majority proteins when tested with a panel of monoclonal antibodies. Troponin I also retained its ability to bind to troponin C in the presence of Ca2+. Recombinant CK-BB and CK-MM which were expressed in the soluble fraction of E. coli cell lysates, contained significantly higher endotoxin levels than recombinant CK-MB, myoglobin and cardiac troponin I which were expressed in the form of inclusion bodies.


CONCLUSIONS
Of the three methods tested, Triton X-114 phase separation was the most effective way of removing endotoxin from recombinant proteins.
Liu
S
S
Spectral Diagnostics, Inc., Toronto, ON, Canada.
Tobias
R
R
McClure
S
S
Styba
G
G
Shi
Q
Q
Jackowski
G
G
D001671:Biochemistry / Q000379:methods*; D002021:Buffers; D002846:Chromatography, Affinity; D003402:Creatine Kinase / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D004731:Endotoxins / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D005069:Evaluation Studies as Topic; D005782:Gels; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D007527:Isoenzymes; D009211:Myoglobin / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000737:chemistry; D011112:Polymyxin B / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D019210:Troponin I / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D002021:Buffers; D004731:Endotoxins; D005782:Gels; D007527:Isoenzymes; D009211:Myoglobin; D011994:Recombinant Proteins; D019210:Troponin I; D011092:Polyethylene Glycols; D006639:Histidine; D017830:Octoxynol; C010615:Nonidet P-40; D003402:Creatine Kinase; D011112:Polymyxin B