Three-step purification of lipopolysaccharide-free, polyhistidine-tagged recombinant antigens of Mycobacterium tuberculosis.
Previous work has shown that the study of host immune responses against Mycobacterium tuberculosis, the causative agent of tuberculosis, requires the availability of multiple mycobacterial antigens. Since purification of protein from M. tuberculosis cells is extremely cumbersome, we developed a protocol for purifying milligram amounts of ten recombinant antigens of M. tuberculosis from E. coli cells. Purified proteins were immunologically active and free of contaminants that confound interpretation of cell-based immunological assays. The method utilizes a three-step purification protocol consisting of immobilized metal-chelate affinity chromatography, size exclusion chromatography and anion-exchange chromatography. The first two chromatographic steps yielded recombinant protein free of protein contaminants, while the third step (anion-exchange chromatography) efficiently removed E. coli lipopolysaccharide, a potent polyclonal activator of lymphoid cells. The recombinant proteins were immunologically indistinguishable from their native (i.e., purified from M. tuberculosis) counterparts. Thus the method provides a way to utilize recombinant proteins for immunological analyses that require highly purified antigens.
Public Health Research Institute, New York, NY 10016, USA.
R
R
Colangeli
A
A
Heijbel
A M
AM
Williams
C
C
Manca
J
J
Chan
K
K
Lyashchenko
M L
ML
Gennaro
D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry / Q000302:isolation & purification*; D002374:Catalase / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006168:Guinea Pigs; D006639:Histidine; D008070:Lipopolysaccharides / Q000737:chemistry; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D010455:Peptides / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification*
D000942:Antigens, Bacterial; D008070:Lipopolysaccharides; D010455:Peptides; D011994:Recombinant Proteins; C033223:polyhistidine; D006639:Histidine; D002374:Catalase