RESUMO
The P1 position of protein inhibitors and oligopeptide substrates determines, to a large extent, association energy with many serine proteinases. To test the agreement of phage display selection with the existing thermodynamic data, a small library of all 20 P1 mutants of basic pancreatic trypsin inhibitor (BPTI) was created, fused to protein III, and displayed on the surface of M13 phage. The wild type of displayed inhibitor monovalently and strongly inhibited trypsin with an association constant of Ka = 3 x 10(11) M(-1). The library was applied to select BPTI variants active against five serine proteinases of different specificity (bovine trypsin and chymotrypsin, human leukocyte and porcine pancreatic elastases, human azurocidin). The results of enrichment with four proteinases agreed well with the available thermodynamic data. In the case of azurocidin, the phage display selection allowed determination of the P1 specificity of this protein with the following frequencies for selected P1 variants: 43% Lys, 36% Leu, 7% Met, 7% Thr, 7% Gln.
Assuntos
Aprotinina/genética , Aprotinina/farmacologia , Bacteriófago M13/genética , Mutação , Serina Endopeptidases/efeitos dos fármacos , Inibidores de Serina Proteinase/genética , Inibidores de Serina Proteinase/farmacologia , Sequência de Aminoácidos , Animais , Aprotinina/biossíntese , Bovinos , Humanos , Cinética , Mutagênese Sítio-Dirigida , Biblioteca de Peptídeos , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/biossíntese , Relação Estrutura-Atividade , Especificidade por Substrato , Suínos , TermodinâmicaRESUMO
In recent years the phage display approach has become an increasingly popular method in protein research. This method enables the presentation of large peptide and protein libraries on the surface of phage particles from which molecules of desired functional property(ies) can be rapidly selected. The great advantage of this method is a direct linkage between an observed phenotype and encapsulated genotype, which allows fast determination of selected sequences. The phage display approach is a powerful tool in generating highly potent biomolecules, including: search for specific antibodies, determining enzyme specificity, exploring protein-protein and protein-DNA interactions, minimizing proteins, introducing new functions into different protein scaffolds, and searching sequence space of protein folding. In this article many examples are given to illustrate that this technique can be used in different fields of protein science. The phage display has a potential of the natural evolution and its possibilities are far beyond rational prediction. Assuming that we can design the selection agents and conditions we should be able to engineer any desired protein function or feature.