A system for stable indirect immobilization of multimeric recombinant proteins

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Current ELISA and phage display methods often immobilize proteins via passive adsorption, leading to denaturation and reduced interaction efficiency.
• Denaturation compromises ligand-receptor interactions dependent on native protein structures.
• Indirect immobilization strategies can prevent protein denaturation during solid-phase attachment.

Purpose of the Study:

• To develop an efficient and stable indirect immobilization system for diverse recombinant multimeric proteins.
• To enable the use of immobilized proteins in their native, biologically active conformation for assays.

Main Methods:

• Constructed a novel vector (pJuFoexpress) for expressing Jun/Fos leucine zipper-linked multimeric proteins.
• Purified proteins using Ni+ affinity chromatography with a 6xHis-ABP fusion tag.
• Exploited the high affinity of albumin binding protein (ABP) for rat serum albumin (RSA) for indirect immobilization.

Main Results:

• The RSA-ABP system demonstrated 10-1000 times greater efficiency in binding assays compared to other immobilization methods.
• Successfully screened and enriched IgG Fc DNA fragments from a phage library using the RSA-ABP system and a staphylococcal protein A bait.
• The pJuFoexpress vector facilitates production and purification of stable, multimeric protein complexes.

Conclusions:

• The pJuFoexpress vector enables indirect, non-chemical immobilization of recombinant proteins, preserving native structure and biological activity.
• This novel system provides stable protein display suitable for enzyme-linked immunosorbent assays (ELISA) and phage display applications.
• The developed method offers a significant improvement for applications requiring native protein conformations.