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To simplify the protein purification process, we developed a novel one-step purification method in which the recombinant protein can be cleaved directly from the Escherichia coli cell surface. This method involves fusion of the target protein to the C-terminus of a LOS tag comprising a surface anchor protein (Lpp-OmpA) and a sequence-specific nickel-assisted cleavage (SNAC)-tag. The LOS tag facilitates the anchoring of the target protein to the outer membrane of E. coli cells and its separation from the cell membrane through Ni2+ cleav