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Evaluation of cation exchange chromatography for the isolation of M glycoprotein from histoplasmin
R M Zancopé-Oliveira1, S L Bragg, S F Hurst
1Laboratório de Micologia, Hospital Evandro Chagas, Fundaçao Oswaldo Cruz, Rio de Janeiro, Brazil.
Abstract:
Cation exchange chromatography was evaluated to purify the M antigen from histoplasmin (HMIN). Two H and M antigen-containing fractions, soluble (S) and precipitate (PP), resulted from the initial 0.025 M, pH 3.5 citrate buffer dialysis step. The PP fraction contained 62% of the M antigen activity and was resolubilized. Both fractions were chromatographed on CM Sepharose CL-6B. Polysaccharide C antigen was abundant in the S fraction and most of it did not bind to CM Sepharose. M antigen-enriched fractions were eluted with 0.5 M NaCl. Re-chromatography of the relevant S fraction (S-II) and PP fraction (PP-II) by linear gradient fast protein liquid chromatography (FPLC) removed protein and C impurities. M antigen purified by FPLC from the PP-II fraction was depleted of other antigens when Western blots were probed with anti-M, anti-H and anti-C monoclonal antibodies (Mabs). M antigen was identified as a 94 kDa glycoprotein containing a specific-protein epitope and an epitope that reacted with a Mab against the polysaccharide C antigen. M antigen can be purified from HMIN by tandem cation exchange chromatography of the precipitable fraction on an open CM Sepharose CL-6B column followed by linear gradient FPLC.
Insights
This study details a new method for purifying M antigen from histoplasmin (HMIN) using cation exchange chromatography. This technique effectively isolates M antigen, a 94 kDa glycoprotein, for further research.
Area of Science:
- Immunology
- Biochemistry
- Chromatography
Background:
- Histoplasmin (HMIN) contains multiple antigens, including M, H, and C.
- Purification of specific antigens like M antigen is crucial for diagnostic and research applications.
Purpose of the Study:
- To develop and evaluate a cation exchange chromatography method for purifying M antigen from HMIN.
- To characterize the purified M antigen and assess its purity.
Main Methods:
- Initial fractionation of HMIN using citrate buffer dialysis into soluble (S) and precipitate (PP) fractions.
- Chromatography on CM Sepharose CL-6B and subsequent re-chromatography using fast protein liquid chromatography (FPLC).
- Western blot analysis using monoclonal antibodies (Mabs) to identify and characterize M antigen.
Main Results:
- The precipitate fraction (PP) contained 62% of M antigen activity.
- Tandem cation exchange chromatography effectively removed protein and C antigen impurities.
- Purified M antigen was identified as a 94 kDa glycoprotein with specific epitopes.
Conclusions:
- Cation exchange chromatography, particularly tandem FPLC, is an effective method for purifying M antigen from HMIN.
- The purified M antigen possesses both protein and polysaccharide C antigen epitopes.
- This purification strategy enables the isolation of M antigen for further immunological studies.