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An efficient expression, purification and immunodetection system for recombinant gene products
G Baier1, G Baier-Bitterlich, C Couture
1La Jolla Institute for Allergy and Immunology, CA.
Biotechniques
|July 1, 1994
Summary
Researchers developed a modified expression vector, pTag/CMV-neo, for easy purification and detection of recombinant fusion proteins using a novel peptide tag. This tool aids in characterizing proteins expressed from the human cytomegalovirus (CMV) or T7 promoters.
Area of Science:
- Molecular Biology
- Recombinant Protein Expression
Context:
- Mammalian expression vectors are crucial for producing recombinant proteins.
- Current methods may lack efficiency in purification and specific detection.
- The pRc/CMV vector offers gene expression control via CMV or T7 promoters.
Purpose:
- To modify the pRc/CMV mammalian expression vector for enhanced recombinant fusion protein expression.
- To enable simple, single-step affinity purification and specific immunodetection of fusion proteins.
- To introduce a novel peptide tag for improved protein characterization.
Summary:
- A modified mammalian expression vector, pTag/CMV-neo, was engineered from pRc/CMV.
- It incorporates a Kozak consensus ribosome-binding site and a 30-amino acid fusion tag.
- The tag includes a (His)6 metal-binding site for Ni(2+)-chelating resin purification and a p18HIV peptide for immunodetection with monoclonal antibody H902.
Impact:
- Facilitates straightforward purification and specific detection of recombinant fusion proteins.
- The p18HIV epitope and H902 antibody offer a specific tool for protein identification and characterization.
- This modified vector system enhances the utility of expression systems driven by CMV or T7 promoters.