TY - JOUR
T1 - Preparation of Chain-End Clickable Recombinant Protein and its Bio-Orthogonal Modification
AU - Wang, Lin
AU - Jiang, Rui
AU - Wang, Lin
AU - Liu, Yang
AU - Sun, Xue-Long
N1 - 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.
PY - 2016/4/1
Y1 - 2016/4/1
N2 - 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 (TM 456 ), 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 TM 456 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 TM 456 was demonstrated. The third is to add azide functionality to the N-terminal amine of recombinant TM 456 via amidation chemistry, and the fourth is tyrosine selective three-component Mannich reaction to introduce azide functionality to recombinant TM 456 . Overall, SML of recombinant protein affords the highest overall yield for incorporating azide functionality into the C-terminus recombinant TM 456 since the key protein expression step uses natural amino acids. Also, single site modification facilitates the highest TM 456 activity.
AB - 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 (TM 456 ), 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 TM 456 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 TM 456 was demonstrated. The third is to add azide functionality to the N-terminal amine of recombinant TM 456 via amidation chemistry, and the fourth is tyrosine selective three-component Mannich reaction to introduce azide functionality to recombinant TM 456 . Overall, SML of recombinant protein affords the highest overall yield for incorporating azide functionality into the C-terminus recombinant TM 456 since the key protein expression step uses natural amino acids. Also, single site modification facilitates the highest TM 456 activity.
KW - Azide
KW - Bio-orthogonal modification
KW - Copper-free click chemistry
KW - Sortase A
KW - Thrombomodulin
UR - https://engagedscholarship.csuohio.edu/scichem_facpub/292
UR - http://www.sciencedirect.com/science/article/pii/S0045206816300177
U2 - 10.1016/j.bioorg.2016.02.010
DO - 10.1016/j.bioorg.2016.02.010
M3 - Article
VL - 65
JO - Bioorganic Chemistry
JF - Bioorganic Chemistry
ER -