ELP-FUSION TECHNOLOGY FOR BIOPHARMACEUTICALS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 207

FIGURE 14.2 Schematic illustration of ELP-based protein purification strategies by inverse
transition cycling (ITC). (A) Soluble ELPylated target proteins (sELPylated TP) became reversibly
insoluble above T t (iELPylated TP). (B) Excess of free sELPs facilitated co-aggregation of sparse
amounts of sELPylated TP above T t to support purification via ITC. (C) Target protein and a protease
are ELPylated. After cleavage of the ELP-target protein, both ELPylated protease and the free ELPs
are precipitated by ITC. (D) Intein-mediated cleavage of ELPylated target protein. The intein-ELP-
fusion protein was precipitated by ITC after cleavage. (E) ELP-mediated affinity capture (EMAC) for
the purification of target compounds without the need for direct ELP fusion. ELPs were fused to a
capture protein (cELPs) that binds specifically and reversibly to the target compound (e.g., protein,
plasmid-DNA, glycopeptides, or heavy metals). Soluble cELPs complexes with the target compound
(sELPs/TC) can be specifically aggregated above T t to form insoluble cELPs/TC complexes
(icELPs/TC). Source: (A), (B), and (E) are reprinted from Reference [9]. Copyright (2010), with
permission from Elsevier.

co-workers found that ammonium sulfate could substitute
sodium chloride at a fourfold lower concentration to achieve
equivalent ELP precipitation. Moreover, the precipitation
step could be performed at room temperature instead of the
commonly used 37 C,

purification procedure [34]. Although ammonium sulfate
triggered the phase transition at a lower molar concentration,
it should be noted that ammonium sulfate precipitated more
contaminant proteins than sodium chloride. Therefore, more
rounds of ITC were necessary to obtain pure ELP-fusion

allowing a cheaper, gentler