The solvent in CNBr cleavage reactions determines the fragmentation efficiency of ketosteroid isomerase fusion proteins used in the production of recombinant peptides

Abstract

Abnormal fragmentation during cyanogen bromide polypeptide cleavage rarely occurs, although parallel side reactions are known to typically accompany normal cleavage. We have observed that cyanogen bromide cleavage of highly hydrophobic fusion proteins utilized for production of recombinant peptides results in almost complete abolishment of the expected reaction products when the reaction is carried out in 70% trifluoroacetic acid. On the basis of mass spectrometric analysis of the reaction products, we have identified a number of fragments whose origin can be attributed to incomplete fragmentation of the fusion protein, and to unspecific degradation affecting the carrier protein. Substituting the solvent in the reaction media with 70% formic acid or with a matrix composed of 6 M guanidinium hydrochloride in 0.1 M HCl, however, was found to alleviate polypeptide cleavage. We have attributed the poor yields of the CNBr cleavage carried out in 70% TFA to the increased hydrophobicity of our particular fusion proteins, and to the poor solubilizing ability of this reaction medium. We propose the utilization of chaotropic agents in the presence of diluted acids as the preferred cyanogen bromide cleavage medium of fusion proteins in order to maximize cleavage efficiency of hydrophobic sequences and to prevent deleterious degradation and structural modifications of the target peptides.