Three-in-one chromatography-free purification, tag removal, and site-specific modification of recombinant fusion proteins using sortase A and elastin-like polypeptides

Abstract

Applied in tandem, elastin-like polypeptides (ELPs) and the sortase A (SrtA) transpeptidase from Staphylococcus aureus provide a general method for chromatography-free purification of tag-free recombinant proteins and optional, site-specific and homogeneous conjugation of the protein to a small molecule. This system provides an efficient, practical mechanism for generating bioactive proteins and protein-small-molecule combination therapeutics at high yields and purities.

Figure 1

(a) SDS-PAGE of target protein purification by reaction of target protein-ELP and SrtA-ELP fusions. Left to right for each target protein: reaction product, centrifugation pellet, and centrifugation supernatant. (B) SDS-PAGE of ternary fusion reactions. Left to right for each target protein: unreacted ternary fusion, reaction product, centrifugation pellet, and centrifugation supernatant.

Figure 2

(a) Viablility of L929 cells after incubation with TNFα from reaction of TNFα isolated from the binary fusion (), from the ternary fusion (), or from eBioscience () relative to an untreated control. Error bars indicate standard deviations. (b) Native PAGE of commercial TNFα (lane 1) and TNFα from the ternary fusion (lane 2) are comparable and suggest the presence of monomer, dimer, and trimer populations in the lower (most mobile) bands.

Figure 3

(a) SDS-PAGE analysis of TRAIL-ELP reactions run in parallel with triglycine (Gly₃) or triglycine-modified camptothecin (Gly₃-CPT). Shown left to right for each reaction are the raw product, centrifugation pellet, and centrifugation supernatant. (b) Cell death in MDA-MB-231 cells treated with TRAIL (), Gly₃-CPT (), TRAIL-CPT conjugate (), or a mixture of Gly₃-CPT and TRAIL at a 0.8:1 molar ratio. (). For combination treatments, the TRAIL concentration is indicated. The viable cell number is reported relative to an untreated group. Error bars indicate standard deviations.

Scheme 1

(a) Overview of ELP fusion protein purification by inverse transition cycling. (b) Schematic of the reaction catalyzed by SrtA-ELP for recovery of target proteins from binary ELP fusions. (c) Strategy for target recovery by reaction of ternary SrtA-ELP-target protein fusions.