Identification of the cleavage site of protein 4.1.

(A) The structure of human erythrocyte protein 4.1 containing 4 structural domains. Hatched box represents the 21-aa peptide within the 10 kDa spectrin–actin-binding domain. (B) SDS-PAGE analysis of recombinant protein 4.1 digested with FP-2. A segment of protein 4.1 encoding aa 294 to 588 expressed as a His-tagged protein inE coli was bound to Ni-NTA beads (lane 1), digested with 0.2 μM rFP-2, pH 7.5, for 30 minutes at 37°C, and centrifuged to collect beads (lane 2), and bound protein was eluted in minimum volume of a buffer containing 1 M imidazole (lane 3). (C) Amino acid sequence of the 10-kd spectrin–actin-binding domain indicating the position of the cleavage site (arrow). Sequence of 3 peptides is underlined, and position of the 21-aa alternative exon is indicated.