Carboxy-terminal protein sequence analysis

Automated carboxy-terminal (C-terminal) protein sequence analysis enables the direct and unambiguous confirmation of the C-terminal sequence of native and expressed proteins, the detection and characterization of protein processing at the C-terminus, the identification of post-translational proteolytic cleavages, and partial sequence information on amino-terminally blocked protein samples. In order for C-terminal sequence analysis to be of immediate benefit, each of the 20 common amino acid residues must be detectable. Additionally, the scope of typically analyzable protein samples must span a usefully broad molecular weight range and degree of structural complexity. 

Protein chemists have sought a practical, automated carboxy-terminal (C-termi-nal) sequencing method for many years. In the biophannaceutical industry, C-ter-minal sequence information can be essential for the early characterization of recombinant proteins, as well as for routine batch analysis. Several automated chemical degradation schemes, analogous in principle to the Edman method for amino-terminal (N-terminal) sequencing, have been proposed (1,2). In 1992, we introduced a novel alkylation method for C-terminal protein sequencing (3,4). 

Indeed, homologous regions of all of the a-actinin protein domains can be found within the sequences of a- and /3-spectrin. For example, the amino and carboxy terminal regions of a-actinin resemble the N-terminus of /3-spectrin and the C-terminus of a-spectrin, respectively (Byers et al, 1989 Dubreuil et al, 1989). Phylogenetic analysis shows a common ancestor for the first repeat of a-actinin and the first repeat of /3-spectrin. Similarly, each of the remaining repeats in a-actinin (2—4) correspond to repeats 1 and 2 of /3-spectrin and repeats 19 and 20 of a-spectrin respectively (Fig. 2). This may have relevance for the function of these repeats in the dimerization of these proteins (Pascual et al, 1997). It is the similarity between these regions of a-actinin, the spectrins, and the simpler domain organization of a-actinin that have led to the hypothesis that these two protein families have evolved from an a-actinin-like precursor. 

Recombinant human interleukin-2 (rIL-2, previously known as T-cell growth factor) was expressed in E. coli (7). During the purification process of rIL-2 by reversed-phase HPLC, another higher molecular weight form of this protein was also isolated, known as HMW rIL-2 (8). The purification process of HMW rIL-2 involved two chromatography steps and utilized a Bakerbond Carboxy-Sulfon (CS) column. Approximately 2 nmol each of rIL-2 and HMW rIL-2 were immobilized using ProSpin cartridges for C-terminal sequence analysis. 

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