Effective Isolation of Mpb64 From a Large Volume of Culture Filtrate of Mycobacterium Bovis BCG Tokyo

Abstract

SUMMARY: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 iso lated MPB64 was tested by immunoblotting with anti-MPB64 antibody. The po tency 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.15

Th1 AdjuvantN-Acetyl-d-Glucosamine Polymer Up-Regulates Th1 Immunity but Down-Regulates Th2 Immunity against a Mycobacterial Protein (MPB-59) in Interleukin-10-Knockout and Wild-Type Mice

“…MPB-59 (30 kDa) was prepared from culture filtrates of M. bovis BCG Tokyo 172 as described previously (19). The bacteria were cultured in Sauton synthetic medium at 37°C without aeration for 8 days.…”

“…with a molecular weight 5,000 cutoff membrane (YM-3; Amicon, Beverly, Mass.). Proteins were further concentrated with 60% saturated ammonium sulfate and fractionated high-pressure liquid chromatography (i) affinity chromatography with phenyl Sepharose CL-4B, (ii) DEAE Sepharose CL-6B ion exchange, (iii) Sephacryl S200 HR gel filtration, and (iv) re-ion-exchange with DEAE Sepharose CL-6B (all from Pharmacia LKB, Uppsala, Sweden) (19). Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis with 10 g of purified MPB-59 protein, a single 30-kDa band was stained by silver (data not shown).…”

Diagnosis of active tuberculosis using MPB64, a specific antigen of Mycobacterium bovis

“…Genetic analysis of various TB proteins has confirmed that MPB64 is identical to MPT64, a protein produced by M. tuberculosis . Non‐tuberculous mycobacteria do not produce MPB64; it is specifically secreted by M. tuberculosis complex (17–21). MPB64 was first isolated by Harboe and Nagai in 1986, whereas Li and colleagues identified it as a secreted protein specific to tuberculous mycobacteria in 1993 (7, 3).…”

Detection of Active Tuberculosis by an MPB-64 Transdermal Patch: A Field Study

“…The protein was isolated from a culture ltrate of M. bovis BCG Tokyo, an 8-d culture, and puri ed according to published techniques (5). The purity of the protein was con rmed by silver staining.…”