Sequencing, N-terminal

N-terminal sequencing of the first 20-30 amino acid residues of the protein product has become a popular quality control test for finished biopharmaceutical products. The technique is useful, as it  

Analogous techniques facilitating sequencing from a polypeptide s C-terminus remain to be satisfactorily developed. The enzyme carboxypeptidase C sequentially removes amino acids from the C-terminus, but often only removes the first few such amino acids. Furthermore, the rate at which it hydrolyses bonds can vary, depending on what amino acids have contributed to bond formation. Chemical approaches based on principles similar to the Edman procedure have been attempted. However, poor yields of derivatized product and the occurrence of side reactions have prevented widespread acceptance of this method. 

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Edman degradation (Section 26.6) A method for N-terminal sequencing of peptide chains by treatment with Af-phenylisothiocyanate. 

Recently, a gene coding for a novel pectin methylesterase, has been cloned (19). This gene, pemB, codes for a 433 amino add protein induding a N-terminal sequence of 21 amino adds which presents the characteristics of lipoprotein... 

Up to now, the pectinolytic enzymes of E. chrysanthemi that have been detected were extracellular secreted enzymes (PelA, B, C, D, E, L, exo-Peh and PemA), periplasmic (exo-Pel), or cytoplasmic (OGL) proteins (1, 5). In contrast, PemB is an outer membrane pectinolytic enzyme. To our knowledge it is the first pectinase characterised as a membrane protein. We presented several lines of evidence showing that PemB is a lipoprotein (i) Its N-terminal sequence has the characteristics of lipoprotein signal sequences, (ii) PemB is synthesised as a high molecular weight precursor processed into a lower molecular weight mature form, (iii) Palmitate, the most prevalent fatty acid in bacterial lipoproteins (12), is incorporated into PemB. 

Hall et al. [62] identified in a separate study the same glycoprotein in H,K-ATPase vesicles isolated from porcine gastric mucosa. A stoichiometric ratio of 1.2 1.0 was found for the deglycosylated protein (35 kDa)/catalytic 94-kDa protein. Furthermore, compelling evidence that this glycoprotein is the H,K-ATPase p subunit was provided by N-terminal sequence analysis of three protease V8-obtained peptides of the 35-kDa core protein. These peptides showed 30% and 45% homology with the Na,K-ATPase pi and pi subunit, respectively. 

The molecular weights of all SERCA-type Ca " transport ATPases are in the range of 100-110 kDa. Their N-terminal sequences are similar Met-Glu-X(Ala, Asn, Glu, Asp)-X (Ala, Gly, He). The Met-Glu-X-X sequence serves as a signal for the acetylation of N-terminal methionine both in soluble and in membrane proteins [71,72]. 

Table 3.1 for the putative carriers in saliva, urine and nasal mucus the N-terminal sequences are highlighted against the pig salivary protein. All lipocalins or similar uncharacterised ( 20kDa) fractions with biological activity are extracellular, often found in quantity, 5 g/ml protein in male mouse urine and with a relatively high negative charge. 

The N-terminal sequence of one peptide from the 35 kDa zone of H-gal-GP showed some homology to cathepsin B-like cysteine proteases. Molecular cloning has also identified a thrombospondin homologue associated with the diffusely staining region between zones A and B, a galectin associated with zone D (Newlands et al., 1999) and a low molecular weight (approximately 13 kDa) cysteine protease inhibitor, cystatin. 

Fig. 13.5. Hydrophobicity analysis of the predicted amino acid sequence from a metalloprotease component of H-gal-GP (MEP3) showing two potential transmembrane domains (indicated by open arrows). B41 and B47 indicate the relative positions of two N-terminal sequences determined from bands present when H-gal-GP is reduced.

Christie, J.F., Dunbar, B. and Kennedy, M.W. (1993) The ABA-1 allergen of the nematode Ascaris suum. epitope stability, mass spectrometry, and N-terminal sequence comparison with its homologue in Toxocara canis. Clinical and Experimental Immunology 92, 125-132. 

Application of the analytical techniques discussed thus far focuses upon detection of proteinaceous impurities. A variety of additional tests are undertaken that focus upon the active substance itself. These tests aim to confirm that the presumed active substance observed by electrophoresis, HPLC, etc. is indeed the active substance, and that its primary sequence (and, to a lesser extent, higher orders of structure) conform to licensed product specification. Tests performed to verify the product identity include amino acid analysis, peptide mapping, N-terminal sequencing and spectrophotometric analyses. 

N-terminal sequencing is normally undertaken by Edman degradation (Figure 7.5). Although this technique was developed in the 1950s, advances in analytical methodologies now facilitate fast and automated determination of up to the first 100 amino acids from the N-terminus of most proteins, and usually requires a sample size of less than 1 umol to do so (Figure 7.6). 

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