MRNA from sc

mRNA

Fig. 35.17. The mRNA display method.

mRNA

peptide fusion

Fig. 35.17. The mRNA display method.

acids can be utilized in the peptide combinatorial libraries through the use of suppressor codons or other strategies [139-142].

Perhaps the most powerful method for in vitro selection of peptide combinatorial libraries is mRNA display, which works by utilizing direct covalent attachment of each member of the peptide combinatorial library to its encoding mRNA (Fig. 35.17) [143, 144]. Such covalent constructs are formed by in vitro translation of 3'-puromycin mRNA templates. After mRNA translation, puromycin enters the pep-tidyl transferase site of the ribosome and is covalently attached to the C-terminus of the nascent peptide. The peptide moiety of the resulting mRNA-peptide conjugate can then be selected as described for the other formats.

In a recent example, 20 different peptide aptamers to streptavidin were identified from a combinatorial library of 1013 different 88 amino acid sequences [145]. These aptamers had binding affinities (KD 5-10 nM) that were three orders of magnitude better than a commercially available streptavidin-binding decapeptide (Kd 13-72 mM). Truncation analysis showed that one of these peptides could be reduced to only 38 amino acids, and also indicated that the sequence motif of histidine-proline-glutamine (HPQ) was essential for binding activity (as in other peptides with affinity for streptavidin [146]). Interestingly, all 20 aptamers that were identified were not from the intended reading frame but from the third reading frame that results from a two-base insertion. This outcome is probably because the HPQ motif occurs more frequently in the third reading frame.

Protein Combinatorial Libraries

Protein design and engineering is an attempt to understand the factors involved in creating folded protein structures with specific behaviors, such as thermal stability or specific catalytic activity, and then utilize that understanding to create novel proteins with useful functions. Owing to our currently incomplete knowledge of the relationship between structure and function, however, the full de novo design of a

Amplification/ diversification of selectants

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