Inverse transition cycling purification

Purification of Spider Silk-Elastin Fusion Proteins by Heat Treatment and Inverse Transition Cycling... 

Fig. 11.3 Purification ofSOl-lOOxELP-proteins from transgenic tobacco plants by inverse transition cycling and analysis by SDS-PAGE. 1 15 pg of total soluble leaf protein extracted in raw extract buffer 2 cleared supernatant of original 15 pg total soluble leaf protein after heat treatment (60 min, 95 °C) 3 cleared supernatant of original 300 pg leaf protein after heat treatment 4 cleared supernatant of original 300 pg leaf protein after heat treatment (60 min, 60 °C) with 2 M NaCI 5 redissolved spider silk-elastin protein pellet from original 300 pg of total soluble leaf protein after heat treatment (60 min, 60 °C) with 2 M NaCI.

Further modifications of the ELR-base protein purification approach have been made in order to circumvent some problems related to protein purification when the protein is expressed at ultra-low levels. One of the multiple factors that influence thermosensitive behavior is polymer concentration. Some proteins, and their respective fusion proteins, have the drawback of being expressed at low levels, which have repercussions on inverse transition cycling efficiency. To overcome this problem, the addition of free ELR to the soluble lysate containing the fusion protein has been proposed [126, 132, 133]. Free ELR acts as a co-aggregant that leads not only to a decrease in T, due to the increase in ELR concentration but also to an easier recovery of aggregates thanks to their large size. This ITC variant focused on the addition of excess ELR has allowed the purification of ultra-low concentration ELR fusion constructs, with several such examples having been reported [126, 132, 133]. 

Meyer E, Chilkoti A(2002) Protein purification by inverse transition cycling. In Golemis EA (ed) Protein-protein interactions a molecular cloning manual. Laboratory Press, Cold Spring Harbor... 

Christensen T, Trabbic-Carlson K, Liu W, Chilkoti A (2007) Purification of recombinant proteins from Escherichia coli at low expression levels by inverse transition cycling. Anal Biochem 360 166-168... 

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