CNBr cleavages

Several highly specific chemical methods of proteolysis are available, the most widely used being cyanogen bromide (CNBr) cleavage. CNBr acts upon methio-... 

Chemical cleavage at selective amino acid residues is an alternative method for scission of peptide bonds in target molecules. Cleavage at the Met-Xaa bond is achieved by CNBr, at the Trp-Xaa bond by the tryptophan-directed reagent 3-bromo-3-methyl-2-[(2-nitro-phenyl)sulfanyl]-3//-indole (BNPS-skatole) and at the Asp-Xaa bond by 2% formic acid.123 24 All these reactions are carried out at acidic pH under standard conditions for each reagent. 

Alternatively, cleavage can be done chemically, but here specificity of cleavage at a single defined position is complicated, since most chemical cleavage methods react with single amino acids (Table 5.13). For instance, chemical cleavage at the C-terminal side of methionine residue can be effected using CNBr, but the use of this method is limited, since most proteins are likely to contain internal methionine residues. 

CNBr cleavage is usually almost complete under these conditions, except that Met-Thr bonds are not noticeably cleaved at all. 

Although rather similar conditions are used for CNBr cleavage (70% formic acid instead of 75%), relatively little Asp-Pro cleavage appears to occur at the lower temperature (20°C instead of 37°C). 

Fig. 1 Summary of the data used to establish the complete amino acid sequence of Er-1 mating pheromone. The peptides have been designated and numbered according to the type of digest ana the theoretical order in which they appear in the sequence. Designations are CNBr, cyanogen bromide T, trypsin V8, . aureus V8 protease CT, chymotrypsin. Peptiaes indicated by two numbers connected with a hyphen result from partial cleavage. Residues directly identified by automated Edman degradation and carboxypeptidase Y digestion (CP-Y) are marked by right and left arrows, respectively, residues identified by amino acid composition are indicated by dashed lines. Taken from ref. 13 and reproduced by permission of the American Society of ...

In order to assign the disulfide bonds of these molecules fast atom bombardment mass spectrometry (FABMS) which has been used not only to confirm amino acid sequence data but also to elucidate post-translational modifications of proteins, such as disulfide bonds, has been employed. For this purpose a sample of native Er-2, containing four methionines, was subjected to CNBr cleavage and without further fractionation directly... 

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.

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