Recombinant Fibronectin Fragment Production

We used the human cDNA encoding the FNIII7-10 fragment and subcloned in the expression vector pET-15b (Takahashi et al., 2007 (link)). To generate the FNIII7-10^syn we mutated by site-directed mutagenesis the two arginines in the synergy sequence: DRVPHSRN>DAVPHSAN. We performed two rounds of PCR using the following primers: 5´-GATGCGGTGCCCCACTCTCGGAAT-3´ (forward) and 5´-GATGCGGTGCCCCACTCTGCGAAT-3´ (forward) and the complementary reverse primers. The expression of recombinant FN fragments was done in the E. coli strain Rosetta T1R. Purification was performed using TALON Metal Affinity chromatography (Clontech, Saint Germain en Laye, France). Finally the protein was obtained by gel filtration chromatography using Superdex 200 10/300 GL columns (GE Healthcare) and Superdex Size Exclusion Media (GE Healthcare, Valencia, Spain) and eluted in PBS.