Abstract
The development of novel methods for highly efficient protein purification remains a research focus in the biotechnology field because conventional purification approaches, including affinity purification, gel filtration, and ion-exchange chromatography, require complex manipulation steps and are costly. Here, we describe a simple and rapid protein purification strategy in which the SUMO tag and Ulp1 protease are surface-displayed separately on Escherichia coli cells. After protein induction, the cells are harvested, resuspended in cleavage buffer, and incubated together for cleavage. In this approach, the surface-displayed Ulp1 cleaves the membrane-anchored SUMO fusion protein, resulting in the release of the target protein from the C-terminal of SUMO into the solution. The bacterial cells harboring SUMO and Ulp1 on their surfaces can be easily removed by centrifugation. To evaluate the purification method, we used red fluorescent protein (mCherry). Purified mCherry protein (7.72 ± 1.05 mg from 1 L of bacterial culture) was obtained after only 30 min of incubation. The protein purity was higher than 80%, and could be further improved (> 90%) by simple ultrafiltration. This study offers a promising and simple strategy for the purification of recombinant protein in its native form that requires only cleavage and centrifugation steps.
Highlights
Proteins are generally produced in heterologous systems because it is difficult to achieve satisfactory yields from natural sources
Zhou et al AMB Expr (2020) 10:65 purification and overcome the disadvantages of the above approaches, we developed a simple and rapid purification strategy based on the cleavage of a SUMO-fused recombinant protein by Ulp1 protease displayed on the surface of Escherichia coli (E. coli) cells
Construction of vectors for surface expression of SUMO‐fused recombinant protein and Ulp1 protease on E. coli cells Various bacterial surface-display systems have been developed for wide applications (Schuurmann et al 2014)
Summary
Proteins are generally produced in heterologous systems because it is difficult to achieve satisfactory yields from natural sources. Zhou et al AMB Expr (2020) 10:65 purification and overcome the disadvantages of the above approaches, we developed a simple and rapid purification strategy based on the cleavage of a SUMO-fused recombinant protein by Ulp protease displayed on the surface of Escherichia coli (E. coli) cells. The SUMO tag can be cleaved from the chimeric protein by Ulp protease, which recognizes the SUMO tertiary structure, to generate the native protein without any redundant amino acids (Li et al 2018). In light of the high efficiency of the SUMO-Ulp system in protein purification, we constructed two different vectors for the expression of SUMO-fused recombinant protein and Ulp protease on the surfaces of E. coli cells. The surface-displayed Ulp cleaves the N-terminal SUMO fusion protein, releasing the native target protein in the buffer solution.
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