Preparation of Chain-End Clickable Recombinant Protein and its Bio-Orthogonal Modification
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.
Wang, L.; Jiang, R.; Wang, L.; Liu, Y.; Sun, X. Preparation of chain-end clickable recombinant protein and its bio-orthogonal modification. Bioorg. Chem. 2016, 65, 159-166.
This work was supported by grants from the American Heart Association (AHA) (14GRANT20290002, X.-L. Sun), NIH (1R01HL102604-04, X.-L. Sun), National Science Foundation (CHE-1126384, X.-L. Sun), Faculty Research Fund from the Center for Gene Regulation in Health and Disease (GRHD) (X.-L. Sun) at Cleveland State University supported by Ohio Department of Development (ODOD).