Protein identity sequence-based

Cell envelopes in numerous bacteria and archaea are covered by a porous layer of proteins. Moreover, for the majority of bacteria this proteinaceous surface layer is de facto composed from numerous identical protein subunits with in the range of 25-200 kDa, and with a copy numbers exceeding 5 x 10 subunits (Sleytr and Messner 1983), thus making them an attractive target for extraction (see Section Surface Layer Proteins ) and sequence-based discrimination of microbial strains. 

This procedure led to a substitution matrix for aligning disordered protein that was different from the commonly used substitution matrices, such as BLOSUM62 (Fig. 4). The matrix for disordered protein is generally better than order-based matrices for aligning disordered proteins whose sequence identities are between 20 and 50%. These results indicate that disordered and ordered protein can be distinguished by their patterns of evolutionary change. 

The translation of the sequence of the cDNA encoding deacetylvindoline 4-O-acetyltransferase compared to other putative plant acetyltransferases revealed a conserved region near the carboxy terminus of the proteins. This sequence was used to design a degenerate antisense oligodeoxynucleotide primer for PCR. The sense primer was based upon an internal peptide sequence of salutaridinol 7-0-acetyltransferase. This approach finally yielded a partial cDNA that encoded salutaridinol 7-O-acetyltransferase. The full-length clone was obtained by RACE-PCR and was functionally expressed in S. frugiperda Sf9 cells.28 The amino acid sequence of salutaridinol 7-O-acetyltransferase is most similar (37% identity) to that of deacetylvindoline acetyltransferase of C. roseus.27... 

Fig. 5. Platform sequence alignments of the MHC class I-like ligands of NKG2D. Sequences of MIC-A and -B, the ULBPs and the RAE-ls have been aligned, divided by family and domain, using CLUSTALW (Thompson et al, 1994). Note that the alignments across families are only very approximate at these levels of sequence identity. Sequences have been numbered from the initiator methionine in the leader peptide, but only the residues in the mature proteins have been shown. Cysteines have been highlighted, and disulhde bond partners have been indicated with matching symbols (, f). For the MIC sequences, allelic substitutions have been indicated by the additional residues shown below the sequences (deletions are indicated with an X ). Diamonds below the sequences indicate NKC2D contact positions, based on the known complex structures (MIC-A 001, ULBPS, and RAE-1/5).

A typical protein evolution experiment starts with an initial protein sequence. Tliis sequence is then copied to a large number of identical sequences. All of these sequences are evolved, or mutated, in parallel. After one round of mutation events, the proteins are screened for figure of merit. This screening step is typically the rate-Umiting step, and so the efficiency of this step determines how many proteins can be evolved in parallel. For typical figures of merit, 10,000 proteins can be screened in a day. If selection, that is, use of a screen based upon whether an organism lives or dies, were performed instead, lO -lO proteins could be screened in a day. Selection is a special case, however, and so the more conservative case of screening 10,000 proteins per day is considered. 

For most chemokines, which contain four cysteines that form two conserved disulfide bonds, a measured mass to charge ratio will be four mass units less than the mass predicted based on protein sequence. Hence, measurement of a recombinant chemokine s mass to charge ratio using a sensitive and accurate mass spectrometer can confirm not only protein identity but also disulfide oxidation as seen in Fig. 4. 

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