Tandem Affinity Purification of Virus-Host Protein Complexes via the Strep/FLAG (SF)-TAP Tag

Area of Science:

• Biochemistry
• Molecular Biology
• Virology

Background:

• Isolating native multiprotein complexes is crucial for understanding viral protein interactions.
• Traditional tandem affinity purification (TAP) often requires protease cleavage steps, which can affect complex integrity.
• The Strep/FLAG-TAP (SF) tag offers a streamlined alternative for protein complex isolation.

Purpose of the Study:

• To describe the mechanisms and experimental procedures for tandem affinity purification using the SF tag.
• To highlight the advantages of the SF tag system for isolating viral-host protein interactions.
• To explore innovations and applications of the SF tag beyond simple purification.

Main Methods:

• Utilizing the SF tag, a modified TAP system incorporating two StrepII and one FLAG tag.
• Performing single-step or tandem purification under mild conditions, eliminating the need for tobacco etch virus (TEV) protease cleavage.
• Applying the method in eukaryotic systems, particularly mammalian cells, to preserve complex stability.

Main Results:

• The SF tag facilitates efficient purification of protein complexes with high purity and minimal nonspecific elution.
• The streamlined process improves the overall efficiency of isolating multiprotein complexes.
• The method demonstrates excellent preservation of complex stability and reduced contamination in mammalian cells.

Conclusions:

• The SF tag system provides an efficient and robust method for isolating native multiprotein complexes, especially for studying viral protein interactions.
• This technique simplifies purification protocols and enhances the quality of isolated complexes.
• Innovations in the SF tag system expand its utility for both protein complex isolation and subsequent functional studies.