Protein production

To produce protein for fiber production, 500 mL of M199+1% FBS and 50 μg/mL G418 was seeded with 5 mL of culture at max density in a 2 L shake flask. After one week of growth at 26°C and 150 rpm, cell cultures were centrifuged at 3500 rpm (~2000 xg) for 15 minutes to pellet the cells. The liquid supernatant was then run through a column containing 1 mL of Ni beads (Roche Product# 05893682001) by gravity flow, the column was washed and bound protein eluted with elution buffer as directed by Roche protocol. Aliquots of elutions were kept for SDS-PAGE analysis and the remainder of each elution could be dialyzed against 5 mM ammonium bicarbonate. The samples, dialyzed or not, were then frozen at -80°C and subsequently lyophilized to powder (0.08 mBarr, -40°C; Labconco FreeZone 2.5).