Skip to content
Login Register

#TAP

3 public questions tagged with this topic.

Protein Sorting and Trafficking

Which of the following is NOT involved in Ran-independent nuclear export of mRNA?

Export of bulk messenger ribonucleoproteins uses machinery fundamentally separate from Ran-dependent protein and small RNA transport. After capping, splicing and polyadenylation, nascent mRNPs acquire TREX complex including THO subcomplex, DEAD-box helicase UAP56 and adaptors Aly/REF and Thp1 that coat transcript and provide binding platform for export receptor. Heterodimer NXF1-NXT1, called TAP-p15 in metazoans, directly contacts FG repeats of central nucleoporins via its NTF2-like and UBA domains to drive translocation without karyopherins or Ran gradient. Directionality and release do not involve Ran but ATP hydrolysis. On cytoplasmic filaments, DEAD-box helicase Dbp5, known as DDX19 in humans, activated by Gle1 bound to inositol hexakisphosphate, hydrolyzes ATP to remove NXF1, Aly and other nuclear factors, remodeling particle and preventing back-sliding into nucleus. Importin-beta, essential for classical NLS import and leucine-rich export, is not part of this complex. Therefore inhibition of Ran cycle or leptomycin B blockade of CRM1 leaves bulk poly(A) export largely unaffected, while NXF1 depletion arrests mRNA export, illustrating Ran independence of messenger pathway central to gene expression and surveillance of spliced transcripts.

Ref: Stewart M, Science 318: 2007, Nuclear Export of mRNA via NXF1-NXT1 and Dbp5 Helicase.

View Details
Protein-Protein Interaction

Which protease is used in TAP?

Tandem Affinity Purification requires a highly specific, mild protease to release first affinity complex without disrupting interacting partners. Tobacco Etch Virus protease recognizes seven amino acid sequence ENLYFQG and cleaves between Q and G with high specificity under low temperature, physiological pH, and in presence of mild detergents that preserve protein-protein interactions. This orthogonal cleavage site is engineered between Protein A and CBP modules. Trypsin, caspase, and collagenase are more promiscuous or require conditions that would dissociate or degrade complexes, unsuitable for preserving interactome.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

View Details
Protein-Protein Interaction

The first step in TAP is:

TAP workflow is designed for high purity through two orthogonal affinities. Lysate containing TAP-tagged bait first incubates with IgG sepharose, where Protein A domain binds heavy chain efficiently. Unbound proteins are washed extensively. Bound material is then released by TEV protease cleavage that separates Protein A from CBP-bait, ensuring specific elution. Second affinity over calmodulin beads in presence of calcium captures CBP. Calmodulin binding never occurs first, and mass spectrometry follows after final EGTA elution. This order minimizes contaminants before identification.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

View Details