Which enzyme is responsible for breaking peptide bonds in proteins?
Proteases (also called peptidases) break peptide bonds in proteins.
Ref: NCERT Class 11 Biology Chapter 9: Biomolecules Enzymes - Properties Classification and Cofactors
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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.
TEV protease in TAP recognizes:
Tobacco Etch Virus protease exhibits stringent specificity for seven amino acid consensus motif Glu-Asn-Leu-Tyr-Phe-Gln-Gly or Ser, cleaving between glutamine and glycine or serine residues. This extended recognition ensures minimal off-target cleavage within mammalian proteins, making it ideal for selective release of protein complexes during Tandem Affinity Purification. Recognition sequence is incorporated between two affinity tags in TAP construct. ATG represents translation initiation codon, calmodulin site binds calcium-dependent calmodulin, and Protein A motif binds immunoglobulin. Seven-residue specificity underlies its utility as biotechnological tool for precise proteolysis.
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.