Engineering Subtilisin into a Fluoride-Triggered Processing Protease Useful for One-Step Protein Purification

Abstract

Subtilisin was engineered into a highly specific, processing protease, and the subtilisin prodomain was coengineered into an optimized recognition sequence. This involved five steps. First, a robust subtilisin mutant was created, which could tolerate the subsequent mutations needed for high specificity. Second, the substrate binding pocket was mutated to increase its sequence selectivity. Third, the subtilisin prodomain was engineered to direct cleavage to the junction of any protein fused to it. Fourth, the active site of subtilisin was engineered to kinetically isolate binding and cleavage reactions. Finally, specific anions were identified to trigger the processing reaction, with fluoride ions being particularly useful. The ability to isolate the binding and processing steps with a triggering mechanism created a protease with a virtual on-off switch. This allowed column-immobilized processing subtilisin to be used as both the affinity ligand and processing protease for one-step purification of proteins. Fusion proteins tagged with the engineered prodomain can be bound to the column and washed free of contaminants. Cleavage can be triggered by the addition of fluoride to release the pure target protein. The column is then regenerated by stripping off the tightly bound prodomain at pH 2.1. Ten proteins have been purified to date by this method.

A minimal sequence code for switching protein structure and function

“…G A and GB variants were cloned into the vector pG58 (16), which encodes an engineered subtilisin prosequence as an N-terminal fusion domain, and the resulting fusion proteins were purified using an affinity-cleavage tag system that we developed (16), essentially as described in ref. 3.…”

“…Minimal medium (14) was used for 15 N and 13 C labeling. Soluble cell extract of prodomain (eXact tag) fusion protein was injected on a 5-mL S189 column at 5 mL/min to allow binding and then washed with 10 column volumes of 100 mM KPO4, pH 7.2 to remove impurities (16). To cleave and elute the purified target protein, 6 mL of 10 mM sodium azide, 100 mM KPO4, pH 7.2 was injected at 0.5 mL/min.…”

The design and characterization of two proteins with 88% sequence identity but different structure and function

“…To facilitate their rapid purification, G A and G B variants were cloned into the vector pG58, which encodes an engineered subtilisin prosequence as the N terminus of the fusion protein, and were purified by using an affinity-cleavage tag system that we developed (35). The system enabled the rapid, standardized purification of mutant proteins, even of low stability.…”

Heterotrimeric G-protein α-Subunit Adopts a “Preactivated” Conformation When Associated with βγ-Subunits

“…Bovine retinas were from W. Lawson Co. (Lincoln, NE). The pG58 expression vector, a fusion vector encoding a modified 77-amino acid prodomain region of subtilisin BPNЈ (proR8FKAM), the pG58-derived expression vector encoding a G t␣ /G i1␣ chimera (Chi6) as a proR8FKAM fusion, and preparation of the S189 subtilisin BPNЈ HiTrap NHS column have been described (30,31). CPK).…”

“…The cell pellet was resuspended in 50 mM Tris-HCl, pH 8.0, containing 50 mM NaCl, 150 M GDP, 5 mM MgCl 2 , 5 mM ␤-mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride, and a protease inhibitor tablet and then disrupted by sonication. The supernatant obtained by centrifugation of the cell lysate at 100,000 ϫ g for 45 min was collected and loaded onto a S189 subtilisin BPNЈ HiTrap NHS column (31). The prodomain-released ChiT was eluted after 12 h in 10 mM Tris-HCl, pH 7.5, containing 100 mM NaCl, 5 mM MgCl 2 , 2.5 mM DTT, 50 M GDP, and 0.1 mM phenylmethylsulfonyl fluoride (Buffer A).…”