Purification and characterization of recombinant mouse interferon-beta

Area of Science:

• Biochemistry
• Immunology
• Molecular Biology

Background:

• Interferons (IFNs) are crucial cytokines in the innate immune response.
• Mouse interferon-beta (MuIFN-beta) plays a significant role in antiviral defense.
• Production of recombinant IFNs is essential for research and therapeutic applications.

Purpose of the Study:

• To purify and characterize recombinant mouse interferon-beta (rMuIFN-beta) produced in Escherichia coli.
• To verify the molecular properties and integrity of the recombinant protein.
• To confirm the identity and post-translational modification status of rMuIFN-beta.

Main Methods:

• Purification of rMuIFN-beta to homogeneity.
• Analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and nonreducing conditions.
• Native polyacrylamide gel electrophoresis (Native PAGE) at pH 4.3.
• Amino acid composition and amino-terminal sequencing.

Main Results:

• Purified rMuIFN-beta showed a single band of Mr 19,900 on SDS-PAGE, consistent with its polypeptide mass.
• A single band was observed on native PAGE, indicating a lack of disulfide-linked dimers.
• Molecular weight and amino acid sequence matched natural MuIFN-beta and predicted cDNA sequence.
• The protein was confirmed to be nonglycosylated.

Conclusions:

• The recombinant mouse interferon-beta produced in E. coli is a homogeneous, nonglycosylated protein.
• The purified rMuIFN-beta exists primarily as a monomeric form.
• These findings validate the recombinant protein as a suitable tool for immunological and biochemical studies.