Enzyme-assisted purification of denatured atelocollagen, using pronase as scavenging agent

Collagen liquid-crystal characteristics can be exploited in advanced applications, like microelectromechanical systems (MEMS), collagen-silica biohybrids or intelligent substrates for tissue engineering. Such applications necessarily require high purity of the collagen colloidal solutions, in terms of macromolecular unitary composition, which is equivalent to almost zero poydispersity. In this work a protocol for the enzyme-assisted removal of non-triple helical polypeptide entities in atelocollagen solutions was conceived, using pronase as scavenger, in the presence of poly(ethyleneglycol) (PEG 10000) as a crowding agent, trimethylamine-N-oxide dihydrate, (TMAO) as kosmotropic agent and anhydrous sodium sulfate as anti-chaotrope salt. Unusually high enzyme concentration (3 : 100 w/w 5 U/mg Pronase : total dry protein) imposes the triple-helix integrity protection, which was achieved by means of protective adjuvants. The adjuvant agents mixture comprising the crowder, the kosmotrope and the anti-chaotropic salt, was formulated according to the Design of Experiments (DOE) principles. The crowding agent represents the key factor in modulating pronase hydrolytic action upon atelocollagen substrate. The optimal adjuvant mixture tested in order to confirm the model validity had the composition: 0.675 PEG, 0.200 TMAO, 0.125 Na2SO4 (mass fractions). The proposed protocol is suitable for purifying medium and large quantities of atelocollagen previously solubilized through an alkali-enzyme technique.