FIG. 5.

Identification of disulfides in CD81 LEL by Lys-C protease digestion and HPLC analysis. (a) Amino acid sequence of purified LEL. Black circles highlight lysines and cysteines. Arrows indicate beginning and end of CD81 LEL. Additional amino acids at the N terminus belong to the joining region between GST and LEL; at the C terminus is the hystidine tail. Letters (A to J) indicate the peptide fragments generated by protease digestion. (b) HPLC analysis of LEL after Lys-C protease digestion. LEL was digested with Lys-C protease under nonreducing conditions, and the generated fragments were separated by RP-HPLC. Each peak was purified and subjected to N-terminal sequencing. Peak 1, fragment I; peak 2, no sequence; peak 3, fragment A; peak 4, fragment D; peak 5, no sequence; peak 6, fragments F, G, and H; peak 7, fragments F, G, and H-I; peak 8, fragments F and G-H. (c) RP-FPLC analysis of peak 7. Peak 7 was purified, reduced with TCEP, and analyzed by RP-HPLC. Peak 9, fragment H-I; peak 10, fragment F; peak 11, fragment G.