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M protein of type 12 Strepto coccus pyogenes. Isolation by electrofocusing and some molecular weight-dependent
Abstract:
A method for the isolation and purification of M protein was developed. Purified cell walls were sonically disrupted, solubilized M protein was precipitated by ammonium sulphate and then electrofocused. Both in this material and in hot acid extracts type-specific trypsin-sensitive antigens with two separately precipitating moieties were found. Evidence is adduced showing that they both belong to the M protein complex. The molecular weight of our purified M protein ranged between 400,000 and 20,000 daltons, giving a peak at 150,000 daltons. The pI of this material was found to be 5.4-5.6. There were marked differences between the behaviour of the low, medium and high molecular weight fractions obtained from purified M protein by gel filtration.
Insights
Researchers developed a new method to isolate and purify M protein. This purification process revealed type-specific antigens within the M protein complex, crucial for understanding its structure and function.
Area of Science:
- Microbiology
- Immunochemistry
- Protein Biochemistry
Background:
- Streptococcal M protein is a key virulence factor.
- Understanding M protein structure is vital for vaccine development.
- Existing methods for M protein isolation are often inefficient.
Purpose of the Study:
- To develop an effective method for isolating and purifying M protein.
- To characterize the isolated M protein and its antigenic properties.
- To investigate the molecular heterogeneity of M protein.
Main Methods:
- Sonic disruption of purified cell walls.
- Ammonium sulfate precipitation of solubilized M protein.
- Isoelectric focusing (pI) and gel filtration for purification and characterization.
- Analysis of type-specific trypsin-sensitive antigens.
Main Results:
- A novel purification method yielding M protein with molecular weights ranging from 20,000 to 400,000 daltons (peak at 150,000 daltons).
- Isoelectric point (pI) of the purified M protein determined to be 5.4-5.6.
- Identification of type-specific, trypsin-sensitive antigens with two precipitating moieties within the M protein complex.
- Demonstrated significant differences in behavior among low, medium, and high molecular weight M protein fractions.
Conclusions:
- The developed method successfully isolates and purifies M protein.
- The M protein complex contains distinct antigenic components.
- Molecular weight heterogeneity of M protein influences its properties.