Qi Huan, Wang Fei, Petrenko Valery A, Liu Aihua
Laboratory for Biosensing, Qingdao Institute of Bioenergy & Bioprocess Technology, and Key Laboratory of Bioenergy, Chinese Academy of Sciences , 189 Songling Road, Qingdao 266101, China.
Anal Chem. 2014 Jun 17;86(12):5844-50. doi: 10.1021/ac501265y. Epub 2014 May 28.
Peptide microarrays evolved recently as a routine analytical implementation in various research areas due to their unique characteristics. However, the immobilization of peptides with high density in each spot during the fabricating process remains a problem, which will affect the performance of the resultant microarray greatly. To respond to this challenge, a novel peptide immobilization method using symmetrical phage carrier was developed in this work. The cellulytic enzyme endoglucanase I (EG I) was used as a model for selection of its specific peptide ligands from the f8/8 landscape library. Three phage monoclones were selected and identified by the specificity array, of which one phage monoclone displaying the fusion peptide EGSDPRMV (phage EGSDPRMV) could bind EG I specifically with highest affinity. Subsequently, the phage EGSDPRMV was used directly to construct peptide microarray. For comparison, major coat protein pVIII fused EG I specific peptide EGSDPRMV (pVIII-fused EGSDPRMV) which was isolated from phage EGSDPRMV was also immobilized by traditional method to fabricate peptide microarray. The fluorescent signal of the phage EGSDPRMV-mediated peptide microarray was more reproducible and about four times higher than the value for pVIII-fused EGSDPRMV-based microarray, suggesting the high efficiency of the proposed phage EGSDPRMV-mediated peptide immobilization method. Further, the phage EGSDPRMV based microarray not only simplified the procedure of microarray construction but also exhibited significantly enhanced sensitivity due to the symmetrical carrier landscape phage, which dramatically increased the density and sterical regularity of immobilized peptides in each spot. Thus, the proposed strategy has the advantages that the immobilizing peptide ligands were not disturbed by their composition and the immobilized peptides were highly regular with free amino-terminal.
由于其独特的特性，肽微阵列最近在各个研究领域作为一种常规分析手段得到了发展。然而，在制造过程中每个斑点中高密度肽的固定仍然是一个问题，这将极大地影响所得微阵列的性能。为应对这一挑战，本研究开发了一种使用对称噬菌体载体的新型肽固定方法。以纤维素酶内切葡聚糖酶I（EG I）为模型，从f8/8景观文库中筛选其特异性肽配体。通过特异性阵列筛选并鉴定出三个噬菌体单克隆，其中一个展示融合肽EGSDPRMV的噬菌体单克隆（噬菌体EGSDPRMV）能以最高亲和力特异性结合EG I。随后，直接使用噬菌体EGSDPRMV构建肽微阵列。作为比较，还通过传统方法固定从噬菌体EGSDPRMV中分离的主要衣壳蛋白pVIII融合EG I特异性肽EGSDPRMV（pVIII-融合EGSDPRMV）来制造肽微阵列。噬菌体EGSDPRMV介导的肽微阵列的荧光信号更具可重复性，比基于pVIII-融合EGSDPRMV的微阵列的值高约四倍，表明所提出的噬菌体EGSDPRMV介导的肽固定方法效率高。此外，基于噬菌体EGSDPRMV的微阵列不仅简化了微阵列构建过程，而且由于对称载体景观噬菌体而表现出显著增强的灵敏度，这极大地提高了每个斑点中固定肽的密度和空间规则性。因此，所提出的策略具有以下优点：固定的肽配体不受其组成的干扰，并且固定的肽具有游离氨基末端且高度规则。
