[Soluble high-expression, purification and bioassay of IGFBP-3]

Area of Science:

• Biotechnology
• Molecular Biology
• Protein Expression

Background:

• Insulin-like Growth Factor Binding Protein 3 (IGFBP3) plays a crucial role in regulating IGF-I bioavailability and action.
• Understanding IGFBP3's function requires reliable methods for its production and characterization.
• Prokaryotic expression systems offer a scalable platform for recombinant protein production.

Purpose of the Study:

• To clone and construct a prokaryotic expression vector for Insulin-like Growth Factor Binding Protein 3 (IGFBP3).
• To express and purify a recombinant fusion protein of IGFBP3 (D-IGFBP3) in E. coli.
• To evaluate the biological activity of the purified D-IGFBP3 in vitro.

Main Methods:

• Cloning of IGFBP3 cDNA and construction of the pET-DsBA-IGFBP3 prokaryotic expression vector.
• Transformation into E. coli BL21 (DE3)plysS and induction of fusion protein expression.
• Purification of D-IGFBP3 using His affinity chromatography and identification via Western blot.

Main Results:

• Successful expression of soluble D-IGFBP3 fusion protein in E. coli.
• Achieved over 95% purity of D-IGFBP3 after His affinity chromatography.
• Demonstrated inhibition of MCF-7 cell growth and in vitro binding with IGF-I by the purified D-IGFBP3.

Conclusions:

• The developed prokaryotic expression system enables efficient production of high-purity recombinant IGFBP3.
• The expressed D-IGFBP3 exhibits biological activity, including growth inhibition and IGF-I binding.
• This recombinant IGFBP3 is a valuable tool for further research into IGF-I signaling pathways and cancer biology.