Recombinant human CIS2 (SOCS2) protein: subcloning, expression, purification, and characterization

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Expression

Background:

• Human CIS2 protein plays a role in cellular signaling.
• Previous methods for obtaining sufficient quantities of active CIS2 were limited.

Purpose of the Study:

• To develop a method for substantial production of biologically active recombinant human CIS2.
• To characterize the recombinant CIS2 protein.

Main Methods:

• Subcloning of 1x myc-tagged human CIS2 cDNA into a pET-29a+ vector for expression in Escherichia coli.
• Isolation from inclusion bodies via solubilization-renaturation and purification using anion-exchange chromatography.
• Assessment of purity and monomeric state using SDS-PAGE and gel-filtration chromatography.
• Verification of biological activity through binding assays with a tyrosine-phosphorylated growth hormone receptor fragment.

Main Results:

• Successfully expressed and purified S-peptide tagged and 1x myc-tagged recombinant human CIS2.
• The recombinant protein was found to be pure and monomeric.
• Demonstrated biological activity by confirming its binding to the target receptor fragment.
• Biochemical, immunological, and molecular comparisons were made between recombinant and eukaryotic-expressed CIS2.

Conclusions:

• This study reports the first substantial production of biologically active recombinant human CIS2.
• The established protocol provides a reliable source for further investigation of CIS2 function.
• The recombinant CIS2 protein is suitable for biochemical and functional studies.