title: Preparation of chain-end clickable recombinant protein and its bio-orthogonal modification.
abstract: 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.
authors.affiliation: Department of Chemistry, Chemical and Biomedical Engineering and Center for Gene Regulation in Health and Disease (GRHD), Cleveland State University, Cleveland, OH, USA.
authors.forename: Lin
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Chemistry, Chemical and Biomedical Engineering and Center for Gene Regulation in Health and Disease (GRHD), Cleveland State University, Cleveland, OH, USA; College of Life and Health Sciences, Northeastern University, Shenyang 110004, PR China.
authors.forename: Rui
authors.initials: R
authors.lastname: Jiang
authors.affiliation: Department of Chemistry, Chemical and Biomedical Engineering and Center for Gene Regulation in Health and Disease (GRHD), Cleveland State University, Cleveland, OH, USA; Department of Medicinal Chemistry, China Medical University, Shenyang, PR China.
authors.forename: Lin
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Chemistry, Chemical and Biomedical Engineering and Center for Gene Regulation in Health and Disease (GRHD), Cleveland State University, Cleveland, OH, USA; Key Laboratory of Structure-Based Drugs Design & Discovery of Ministry of Education, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang, PR China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Chemistry, Chemical and Biomedical Engineering and Center for Gene Regulation in Health and Disease (GRHD), Cleveland State University, Cleveland, OH, USA. Electronic address: x.sun55@csuohio.edu.
authors.forename: Xue-Long
authors.initials: XL
authors.lastname: Sun
mesh_terms: D001386:Azides / Q000737:chemistry / Q000378:metabolism; D057930:Click Chemistry; D004926:Escherichia coli / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D006801:Humans; D008715:Methionine / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D018180:Thrombomodulin / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001386:Azides; D011994:Recombinant Proteins; D018180:Thrombomodulin; D008715:Methionine
keywords: Azide; Bio-orthogonal modification; Copper-free click chemistry; Sortase A; Thrombomodulin