Evaluation of methods used to purify acid-extracted group A streptococcal M protein
Abstract:
The literature includes descriptions of both acid-soluble and acid-insoluble M protein in the preparation of "hot acid-extracted group A streptococcal M protein." We present evidence for the contamination of crude type 1 acid-insoluble M protein. The purification of preparations of crude and partially purified acid-soluble type 1 and type 12 M protein is described. Our quantitative criteria for purification were recovery of M precipitin activity, improvement in specific activity, and removal of carbohydrate. Exclusion of nucleic acid is also discussed. Greater purification in a single passage was found with a carboxymethylcellulose column (with acidic elution) than with hydroxyapatite, diethylaminoethyl-Sephadex, or carboxymethyl-cellulose (with neutral elution) columns or with ammonium sulfate fractional precipitation. Carboxymethylcellulose with acidic elution was found to be a satisfactory standard laboratory procedure for the preparation of purified acid-extracted (acid-soluble) group A streptococcal M protein.
Insights
Purifying acid-extracted M protein from group A Streptococcus is crucial. Carboxymethylcellulose chromatography with acidic elution offers a superior method for obtaining highly purified M protein, removing contaminants like carbohydrates and nucleic acids.
Area of Science:
- Microbiology
- Immunology
- Biochemistry
Background:
- Literature describes both acid-soluble and acid-insoluble M protein in hot acid-extracted group A streptococcal M protein preparations.
- Evidence suggests contamination in crude type 1 acid-insoluble M protein preparations.
Purpose of the Study:
- To describe the purification of crude and partially purified acid-soluble type 1 and type 12 M protein.
- To establish quantitative criteria for M protein purification.
Main Methods:
- Purification of acid-soluble M protein using various chromatographic techniques including carboxymethylcellulose (acidic and neutral elution), hydroxyapatite, and diethylaminoethyl-Sephadex.
- Ammonium sulfate fractional precipitation was also employed.
- Quantitative assessment involved recovery of M precipitin activity, specific activity improvement, and removal of carbohydrate and nucleic acid.
Main Results:
- Carboxymethylcellulose chromatography with acidic elution yielded greater purification in a single passage compared to other methods.
- This method demonstrated superior removal of contaminants like carbohydrate and nucleic acid.
- Quantitative criteria for purification were met effectively.
Conclusions:
- Acid-insoluble M protein can contaminate crude preparations.
- Carboxymethylcellulose chromatography with acidic elution is a satisfactory standard laboratory procedure for preparing purified acid-extracted (acid-soluble) group A streptococcal M protein.
- This method ensures high purity and removal of common contaminants.
