Effective isolation of MPB64 from a large volume of culture filtrate of Mycobacterium bovis BCG Tokyo.
MPB64, a secretory protein of Mycobacterium bovis BCG Tokyo, was isolated from a culture filtrate of the bacteria in Sauton synthetic medium harvested on day 8. The protein was isolated by five steps; (i) concentration of the culture filtrate by cutting the molecules smaller than 5 kDa with the Millipore Pellicon Cassette system, (ii) affinity separation by a Phenyl Sepharose CL-4B column, (iii) separation with a DEAE-Sepharose CL-6B column with 3 M urea, (iv) separation with a Sephacryl S200HR column, and (v) separation with a DEAE-Sepharose column without urea. MPB64 in each fraction was determined by comparing the band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with that of standard MPB64. The specificity of isolated MPB64 was tested by immunoblotting with anti-MPB64 antibody. The potency of isolated MPB64 in eliciting skin reaction in the BCG-sensitized guinea pigs was the same to that of standard MPB64. The method described herein is an improved one for isolating MPB64 from a large volume of culture filtrate of M. bovis BCG Tokyo. The technique should be applicable to isolation of other mycobacterial secretory proteins.
Haga
S
S
Department of Bacteriology, National Institute of Health, Tokyo, Japan.
Kawajiri
K
K
Niinuma
S
S
Honda
I
I
Yamamoto
S
S
Toida
I
I
Nakamura
R M
RM
Nagai
S
S
D000942:Antigens, Bacterial; D001426:Bacterial Proteins / Q000302:isolation & purification*; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D007526:Isoelectric Point; D008970:Molecular Weight; D009163:Mycobacterium bovis / Q000737:chemistry*
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D003470:Culture Media; C080865:MPB64 protein, Mycobacterium