Kiczak L, Koscielska K, Otlewski J, Czerwinski M, Dadlez M
Institute of Biochemistry and Molecular Biology, University of Wroclaw, Poland.
Biol Chem. 1999 Jan;380(1):101-5. doi: 10.1515/BC.1999.014.
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.
蛋白质抑制剂和寡肽底物的P1位置在很大程度上决定了与许多丝氨酸蛋白酶的结合能。为了测试噬菌体展示筛选结果与现有热力学数据的一致性，构建了一个包含碱性胰蛋白酶抑制剂（BPTI）所有20种P1突变体的小型文库，将其与蛋白III融合，并展示在M13噬菌体表面。展示的野生型抑制剂以单价形式强烈抑制胰蛋白酶，结合常数Ka = 3×10¹¹ M⁻¹。该文库用于筛选对五种具有不同特异性的丝氨酸蛋白酶（牛胰蛋白酶和胰凝乳蛋白酶、人白细胞弹性蛋白酶和猪胰弹性蛋白酶、人天青杀素）有活性的BPTI变体。用四种蛋白酶进行富集的结果与现有热力学数据吻合良好。对于天青杀素，噬菌体展示筛选确定了该蛋白的P1特异性，所选P1变体的频率如下：43%为赖氨酸，36%为亮氨酸，7%为甲硫氨酸，7%为苏氨酸，7%为谷氨酰胺。
