Cloning of FKBP to ELP58 (link) and RGD to ELP48 (link) was performed as described previously. pET25b (+) vectors encoding ELP genes were transfected into BLR (DE3) E. coli cells (69053, EMD Millipore, Billerica, MA) and plated on agar with 100 μg/mL ampicillin. A single colony for each construct was inoculated in 50 mL of autoclaved Terrific broth media (12105, Mo Bio Laboratories, Carlsbad, CA) supplemented with 100 μg/mL ampicillin and grown overnight at 37 °C. Starter cultures were then amplified in 3–4 L batches supplemented with 100 μg/ mL ampicillin and allowed to grow for 24 h at 37 °C. Cell lysis and ELP purification was performed using inverse transition cycling (ITC), as described previously.11 ,35 (link) Purified protein in sterile PBS was filtered using 200 nm sterile Acrodisc 25 mm filters (PN 4612, Pall Corporation, Port Washington, NY) and assayed for concentration using Beer–Lambert’s law:
where M is molar concentration; A280 and A350 are absorbance at 280 and 350 nm; respectively; l is the path length (cm); and MEC is the estimated molar extinction coefficient at 280 nm: 1285 M−1 bcm−1 for SI, IS, and ISR; and 11585 M−1 cm−1 for FSI.59 (link) Purified yields were ~50 mg/L of bacterial culture.
where M is molar concentration; A280 and A350 are absorbance at 280 and 350 nm; respectively; l is the path length (cm); and MEC is the estimated molar extinction coefficient at 280 nm: 1285 M−1 bcm−1 for SI, IS, and ISR; and 11585 M−1 cm−1 for FSI.59 (link) Purified yields were ~50 mg/L of bacterial culture.