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biochemistry

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Biochemie

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Creation of the artificial enzyme . The LmrR variant used in this study , referred to as LmrR _ V15pAF , contains a non - canonical paf residue at position 15 inside the hyhobic pore4 . The paf residue can be introduced directly using the dedicated orthogonal translation system26 , but in our experience it is most practical to use the system for incorporation of p - azidophenylalanine ( pAzF ) followed by Staudinger reduction with tris ( 2 - carboxyethyl ) phosphine , which was added to the protein solution after strep - tag affinity purification but before the final dialysis step . Hence , no additional handling steps were required in comparison with direct incorporation of paf . The Lewis acidic site was then introduced through supramolecular self - assembly by combining LmrR _ V15pAF with Cu ( ii ) phen in a buffered solution ( 20 mM mops , 150 mM NaCl , pH 7.0 ) . The binding affinity of Cu ( ii ) - phen to LmrR _ V15pAF was determined by titration through monitoring of the quenching of tryptophan flu

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m-h

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الكيمياء الحيوية