Proteins, cleavage with cyanogen bromide

In the process used by Lilly, insulin is produced in the form of proinsulin as a fusion protein where it is joined to the protein tryptophan synthase via a terminal methionine. The tryptophan synthase is then removed by cleavage with cyanogen bromide. The proinsulin is then converted to insulin via treatment with a mixture of the protease enzymes tiypsin and carbox) eptidase. 

In the treatment of RNase-A with cyanogen bromide, chain cleavage occurs at Met 13. The peptide comprising residues 1-13, where 13 is now homoserine or homoserine lactone rather than methionine, is designated C-peptide (139). This derivative added to S-protein at a molar ratio of 600 1 gave between 50 and 80% of the maximum activity. 

Figure 1 Strategy for cloning a peptide-coding sequence (CDS) as tandem repeats in the vector pET31b. The resulting fusion protein, comprising the ketosteroid isomerase (KSI), peptide repeats, and His-tag, is targeted to inclusion bodies. The fusion protein can be recovered and cleaved, in this case, with cyanogen bromide (CNBr) which acts at the methionine (M) residues allowing further separation of pure peptide from the other fusion components. The cleavage by CNBr results in a C-terminal homoserine lactone (hsl) on each peptide monomer.

Treatment of proteins with cyanogen bromide results in cleavage of the peptide chain COOH-terminal to methionyl residues with concomitant conversion of the methionine to homoserine lactone which is in equilibrium with homoserine. In certain instances (e.g. -Met-Ser-or -Met-Thr-) some of the methionine is converted to homoserine without peptide bond cleavage (see Schroeder et al. 1969). Homoserine and its lactone are also products of the breakdown of the carboxymethylsulfonium salts of methionine ( 2.5.10). 

Conversion of half-cystine residues in proteins and peptides to the S-methyl derivatives is advantageous in subsequent studies of amino acid sequence. Under the usual conditions of acid hydrolysis ( 2.1), S-methylcysteine is recovered in a 90% yield (Heinrikson 1971). The phenylthiohydantoin of S-methylcysteine is readily identified by routine thin layer chromatography procedures (Rochat et al. 1970). With the increasing use of the sequenator, PTH-S-methylcysteine offers a marked advantage over derivatives such as PTH-cysteic acid, or PTFl-carboxymethylcysteine, which have to be identified by special techniques (Edman 1960, 1970). S-methylcysteinyl residues provide a new point of cleavage for cyanogen bromide (5). 

Cleavage of the intact protein with cyanogen bromide yields methionine and a pen-tapeptide. Treating the intact hexapeptide with trypsin yields a dipeptide, which contains tyrosine and glutamate, and a tetrapeptide. When the intact hexapeptide is treated with carboxypeptidase A, a tyrosine residue and a pentapeptide are produced. Bearing in mind that the hexapeptide is isolated from a mouse, write its amino acid sequence, using both three-letter and one-letter abbreviations. 

Cleavage of proteins at methionine with cyanogen bromide yields peptides that have homoserine at the COOH-terminus. Treatment of the peptide with anhydrous trifluoroacetic acid results in a lactone that is sufficiently activated to undergo aminolysis by aliphatic amines (Horn, 1975) or sequencing resins (Wachter et ai, 1975 Horn and Laursen, 1973 Bridgen, 1975) (Fig. 5). In this way, peptides are selectively attached at the COOH-terminus without the need for amino or carboxyl protection. Resins incorporating a )5-diamine structure, such as triethylenetetramine (TETA) polystyrene (Horn and Laursen, 1973) and j8-aminoethylaminopropylglass... 

Also suited for the specific enzymatic hydrolysis of peptide chains is the endoproteinase Glu-C from Staphylococcus aureus V8. It cleaves Glu-X bonds (ammonium carbonate buffer pH 7.8 or ammonium acetate buffer pH 4.0) as well as Glu-X plus Asp-X bonds (phosphate buffer pH 7.8). The most important chemical method for selective cleavage uses cyanogen bromide (BrCN) to attack Met-X-hnkages (Reaction 1.86). Hydrolysis of proteins with strong acids reveals a difference in the rates of hydrolysis of peptide bonds depending on the adjacent amino acid side chain. Bonds involving amino groups of serine and threonine are particularly susceptible to hydrolysis. This effect is due to... 

Spectra to be analyzed via PMF are derived from a protein sample been treated with an enzyme (e.g., trypsin) or other chemical (cyanogen bromide) with specific cleavage activity. The experimental m/z values for each peptide are converted into peptide masses and compared with the theoretical mass... 

Cytochrome c(l-65) and -(66-104), derived by cyanogen bromide cleavage, form a stable complex in which the 65-66 peptide bond spontaneously slowly re-forms to give a protein with full biological activity.1124 ... 

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