Identification of an alpha-1 antitrypsin variant with enhanced specificity for factor XIa by phage display, bacterial expression, and combinatorial mutagenesis
| Autor: | Darian L. Perruzza, Amy Nouanesengsy, Jessica Lapierre, William P. Sheffield, Mostafa Hamada, Varsha Bhakta |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
Proteases congenital hereditary and neonatal diseases and abnormalities Phage display Molecular biology medicine.medical_treatment Science Gene Expression 030204 cardiovascular system & hematology Serpin Biochemistry Article Factor XIa law.invention 03 medical and health sciences 0302 clinical medicine Thrombin law Peptide Library medicine Escherichia coli Humans Multidisciplinary Protease Molecular medicine Chemistry Mutagenesis Kallikrein Recombinant Proteins 030104 developmental biology alpha 1-Antitrypsin Recombinant DNA Medicine medicine.drug |
| Zdroj: | Scientific Reports Scientific Reports, Vol 11, Iss 1, Pp 1-14 (2021) |
| ISSN: | 2045-2322 |
| Popis: | Coagulation Factor XIa (FXIa) is an emerging target for antithrombotic agent development. The M358R variant of the serpin alpha-1 antitrypsin (AAT) inhibits both FXIa and other proteases. Our aim was to enhance the specificity of AAT M358R for FXIa. We randomized two AAT M358R phage display libraries at reactive centre loop positions P13-P8 and P7-P3 and biopanned them with FXIa. A bacterial expression library randomized at P2′-P3′ was also probed. Resulting novel variants were expressed as recombinant proteins in E. coli and their kinetics of FXIa inhibition determined. The most potent FXIa-inhibitory motifs were: P13-P8, HASTGQ; P7-P3, CLEVE; and P2-P3′, PRSTE (respectively, novel residues bolded). Selectivity for FXIa over thrombin was increased up to 34-fold versus AAT M358R for these single motif variants. Combining CLEVE and PRSTE motifs in AAT-RC increased FXIa selectivity for thrombin, factors XIIa, Xa, activated protein C, and kallikrein by 279-, 143-, 63-, 58-, and 36-fold, respectively, versus AAT M358R. AAT-RC lengthened human plasma clotting times less than AAT M358R. AAT-RC rapidly and selectively inhibits FXIa and is worthy of testing in vivo. AAT specificity can be focused on one target protease by selection in phage and bacterial systems coupled with combinatorial mutagenesis. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full text from SpringerLink