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Metal ions-binding T4 lysozyme (mbT4L) - Coggle Diagram
Metal ions-binding T4 lysozyme
(mbT4L)
T4 lysozyme
Lysozyme of enterobacteria phage T4
Also known as endolysin, muramidase
Well folded soluble protein
Replace the flexible intracellular loops of target protein
Enzymatic activity is preserved upon single substitutions of T4L residues
Crystal structure mbT4L
Flexibility between the lobes:
enzymatic activity/ poor crystal quality
Gly/ser linker:
enzymatic inactivity/ limited flexibility/ improved crystallization properties of the mutant protein
Point mutation to Histidine
M77A mutation(Met-> Ala):
prevent the interference by covalent bonds with metal ions and introduced histidines.
Ni binding structure
4 Ni per asymmetric unit
Two Ni formed covalent bonds with
H73, H85, H89 from the same mbT4L
The other two Ni formed with
H76, H80 from one and H82 from the other mbT4L
:check:Not able to solve the entire mbT4L structure using just the anomalous scattering of Ni and SAD
=>
MR-SAD + anomalous scattering can be applied to solve the crystal structure of mbT4L-fused protein
Intramolecular protein purification tag
PI4K2A-trT4L/ -mbT4L
Cysteine-rich motif (-LCCPCCF-) post-translantionally modified with palmitoyl moieties.
Palmitoylated segment
of PI4K2A: perfect target for replacement with T4 lysozyme.
PI4K2A-mbT4L can be freely accessed by immobilized metal ions (such as NiNTA-immobilized nickel ions).
High production of mbT4L
Both constructs expressed well in E. coli with high yields
Yields of chimeric protein:
His-PI4K2AtrT4L < PI4K2A-mbT4L
Concentration of IMD(elution):
His-PI4K2AtrT4L < PI4K2A-mbT4L
Affinity of Ni :
His-PI4K2AtrT4L < PI4K2A-mbT4L
High concentration of IMD is disadvantageous for the target protein, can be modulated by pH, salt concentration, addition of EDTA of the elution buffer, similarly as histidine-tagged proteins
Advantage
Tolerance of changing amino acid sequence
MbT4L is not affected by degradation of disordered N terminus of the mbT4L-fused protein by N-terminal peptidases. Negligible degradation of the purification tag also contributes to higher yields of the purified protein
Solubility
Crystallogenesis
Improves the solubility of the target protein.
May facilitate crystallogenesis through formation of specific metal ions- dependent crystal contacts of mbT4L
Higher Purity
Stronger affinity than the His6-tagged protein, resulting in its higher purity upon purification by the IMAC technique.
MR-SAD
Anomalous scattering
MR-SAD technique and the anomalous scattering from the metal atoms can be applied to solve the crystal structure of a target mbT4L-fused protein
