Sanger Was The First To Determine The Sequence Of A Polypeptide

Mature insulin consists of the 21-residue A chain and the 30-residue B chain linked by disulfide bonds. Frederick Sanger reduced the disulfide bonds (Figure 4-3),

Figure 4-3. Oxidative cleavage of adjacent polypeptide chains linked by disulfide bonds (shaded) by per-formic acid (left) or reductive cleavage by p-mercap-toethanol (right) forms two peptides that contain cysteic acid residues or cysteinyl residues, respectively.

Figure 4-4. Use of SDS-PAGE to observe successive purification of a recombinant protein. The gel was stained with Coomassie blue. Shown are protein standards (lane S) of the indicated mass, crude cell extract (E), high-speed supernatant liquid (H), and the DEAE-Sepharose fraction (D). The recombinant protein has a mass of about 45 kDa.

Figure 4-4. Use of SDS-PAGE to observe successive purification of a recombinant protein. The gel was stained with Coomassie blue. Shown are protein standards (lane S) of the indicated mass, crude cell extract (E), high-speed supernatant liquid (H), and the DEAE-Sepharose fraction (D). The recombinant protein has a mass of about 45 kDa.

Figure 4-5. Two-dimensional IEF-SDS-PAGE. The gel was stained with Coomassie blue. A crude bacterial extract was first subjected to isoelectric focusing (IEF) in a pH 3-10 gradient. The IEF gel was then placed horizontally on the top of an SDS gel, and the proteins then further resolved by SDS-PAGE. Notice the greatly improved resolution of distinct polypeptides relative to ordinary SDS-PAGE gel (Figure 4-4).

separated the A and B chains, and cleaved each chain into smaller peptides using trypsin, chymotrypsin, and pepsin. The resulting peptides were then isolated and treated with acid to hydrolyze peptide bonds and generate peptides with as few as two or three amino acids. Each peptide was reacted with 1-fluoro-2,4-dinitroben-zene (Sanger's reagent), which derivatizes the exposed a-amino group of amino terminal residues. The amino acid content of each peptide was then determined. While the e-amino group of lysine also reacts with Sanger's reagent, amino-terminal lysines can be distinguished from those at other positions because they react with 2 mol of Sanger's reagent. Working backwards to larger fragments enabled Sanger to determine the complete sequence of insulin, an accomplishment for which he received a Nobel Prize in 1958.

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