FIGURE 1.

N-end rule substrate immobilization on a ClpS affinity column and elution with peptides possessing N-degrons. Small columns with 1-ml bed volumes of various AminoLink resins were equilibrated with PBS. Samples were loaded, and the columns were washed at ∼1 ml/min with PBS with and without addition of peptides. GFP-proteins were detected by fluorescence measurements. A, LR-GFP_VENUS (1 mg) was applied to a column cross-linked with ClpS. The column was washed with buffer, and 1 mm FKTA was added to elute the bound LR-GFP_VENUS. B, LR-GFP_VENUS (1 mg) was applied to a control resin prepared by inactivation with Tris buffer in the absence of ClpS. Most of the protein (98%) was recovered in the flow-through fractions. No additional protein emerged after FKTA addition. C, LR-GFP_VENUS (1 mg) was added to a ClpS column. The column was washed with buffer containing 1 mm SLRKGE followed by buffer containing 1 mm LRKGE. D, LR-GFP_VENUS (1 mg) was applied to a column cross-linked to the variant ClpS-D35A,D36A. Most of the protein (>95%) was recovered in the flow-through fractions, and no additional protein was detected after adding FKTA.