Proteins complete sequence analysis

Complete sequence analysis based on cDNA was achieved for osteonectin and SPARC and indicated a disulfide-bonded, four-domain structure for the protein (Fig. 12). Remarkable features of the structure are a cluster of 16 glutamic acid residues at the N terminus and an EF-hand domain located between the last two cysteine residues, both regions having the potential for calcium-binding (Bolander et al., 1988 Engel et al., 1987). Studies with BM-40 demonstrated a reversible change in a helix from 30 to 20% upon removal of calcium. The data indicate the... 

With methods for the quantitative analysis of amino acids to hand, the way was now open for the determination of amino acid sequences. Purified bovine insulin was relatively freely available. On the basis of ultracentrifugal analysis (Gutfreund and Ogston), a molecular weight of 12,000 was assumed—as it later emerged, a factor of 2 too high. One of the advantages from the choice of insulin as the protein to sequence was that tryptophan is absent. A 100% recovery of the amino acids could therefore be obtained easily by simple hydrolysis with HC1. In 1948 Tristram reported the complete amino acid composition of the protein. 

The inability to obtain complete protein sequence analysis of purified bacteriocins has been reason to suspect the presence of N-blocked peptide sequences (34) or lantibiotic residues (14). Recently, Piard et al. (14) have shown from partial sequencing and composition analysis that lacticin 481, a broad spectrum bacteriocin produced by L. lactis 481, also contains lanthionine residues. The early widespread interest in nisin and nisin-producing strains had given the impression that lantibiotics may be characteristic of bacteriocins of lactic acid bacteria. However, recent studies with other LAB bacteriocins suggest that simple peptide bacteriocins may prevail among the LAB. 

Blastocystis hominis is a unicellular anaerobic organism that inhabits the human intestinal tract, and whose metabolism and life cycle are not completely understood. Molecular phylogenetic analysis of the SSU rRNA sequences (Silberman et al. 1996) and protein-coding sequences (Arisue et al. 2002c) show Blastocystis branching within stramenopiles (heterokonts), a group containing a vast diversity of photosynthetic and heterotrophic protists (Fig. 10.2). 

This procedure is ideal for identification of well-resolved, reasonably abundant proteins and for the design of oligonucleotide probes for subsequent cDNA or genomic analysis. It is not a reliable way of obtaining complete protein sequences, however, since many large and hydrophobic peptides are not efficiently retrieved. For full sequence analysis it is necessary to use the fragmentary protein data as a probe for cDNA cloning. 

In theory, Edman degradations could sequence a peptide of any length. In practice, however, the repeated cycles of degradation cause some internal hydrolysis of the peptide, with loss of sample and accumulation of by-products. After about 30 cycles of degradation, further accurate analysis becomes impossible. A small peptide such as bradykinin can be completely determined by Edman degradation, but larger proteins must be broken into smaller fragments (Section 24-9E) before they can be completely sequenced. 

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