کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
1355715 | 1500449 | 2016 | 8 صفحه PDF | دانلود رایگان |
• Four site-selective strategies are applied for incorporating azide functionality into recombinant thrombomodulin.
• Sortase-mediated ligation affords the highest overall yield for incorporating azide functionality into recombinant thrombomodulin.
• Single-site modification facilitates the highest thrombomodulin activity.
• The thrombomodulin-azides were confirmed by fluorescent labeling via click chemistry.
Introducing unique functional group into protein is an attractive approach for site-selective protein modification applications. In this report, we systemically investigated four site-selective strategies to introduce azide functionality into recombinant thrombomodulin (TM456), via direct recombinant expression with unnatural amino acid, chemical, and enzymatic modification for its bio-orthogonal modification application. First, a straightforward recombinant method to express TM456 with azide functionality near C-terminus by replacing methionine with azidohomoanlanine from methionine auxotroph Escherichia coli cell was investigated. Next, a sortase-mediated ligation (SML) method to incorporate azide functionality into the C-terminus of recombinant TM456 was demonstrated. The third is to add azide functionality to the N-terminal amine of recombinant TM456via amidation chemistry, and the fourth is tyrosine selective three-component Mannich reaction to introduce azide functionality to recombinant TM456. Overall, SML of recombinant protein affords the highest overall yield for incorporating azide functionality into the C-terminus recombinant TM456 since the key protein expression step uses natural amino acids. Also, single site modification facilitates the highest TM456 activity.
Four different strategies to introduce azide functionality into recombinant thrombomodulin (TM), site-specifically by recombinant, chemical, and enzymatic methods at either N-terminus or C-terminus for its bio-orthogonal modification application.Figure optionsDownload as PowerPoint slide
Journal: Bioorganic Chemistry - Volume 65, April 2016, Pages 159–166