Production of recombinant Fibronectin tipe III 9-10 domain in bacterial expression system

Fibronectin (Fn) is a glycoprotein that plays important roles in cell adhesion, growth, differentiation and migration by mediating a wide variety of cellular interactions with the extracellular matrix (ECM). Fn usually exists as a dimer composed of two nearly identical 220~250 kDa subunits where each monomer is composed of homologous repeats of three prototypical domains: type I, type II and type III. Fn interacts with many integrins such as α3β1, α5β1, α8β1, αvβ1, αIIββ3, αvβ3, αvβ5, and αvβ6. In previous studies, the specific integrin-recognition sequences involved in cell adhesion have been identified. The best known of these – Arg-Gly-Asp (RGD) sequence – is located in the central cell-binding domain - FnIII10. It is the most important recognition site that can interact with about half of all known integrins. Another important sequence which acts in synergy with the RGD site is - Pro-His-Ser-Arg-Asn (the ‘synergy site’ PHSRN) found in Fn repeat III9, that promotes specific α5β1 integrin binding. Because of the ECM binding properties Fn is a perfect protein for nanofabrication and integrin-mediated immobilization of cells into synthetic scaffolds. Heterologous expressions of large eucaryotic proteins, such as Fn, in bacterial expression system is complicated. Therefore the aim of this study was to establish a recombinant protein production system for the "cell-binding domain" of Fn protein - including FnIII9-10 fragment and to assess the effect of His-tag position on the recombinant peptide purification efficiency. For this purpose, a sequence of the FnIII9-10 fragment was cloned to pLATE bacterial expression vector using a ligation independent cloning system. This vector includes bacteriophage T7 promoter that ensures high yields of expressed proteins. Two constructs including either amino- or carboxy- terminal 6xHis-tag were developed.[...]Biochemijos katedraGamtos mokslų fakultetasLietuvos agrarinių ir miškų mokslų centro Sodininkystės ir daržininkystės institutasVilniaus universitetasVytauto Didžiojo universiteta