Morita Yasutaka, Mamiya Kou, Yamamura Shohei, Tamiya Eiichi
Department of Biological and Environmental Chemistry, School of Humanity-Oriented Science and Engineering, Kinki University, Kayanomori, Iizuka, Fukuoka, Japan.
Biotechnol Prog. 2006 Jul-Aug;22(4):974-8. doi: 10.1021/bp060112f.
The P19 cell is a pluripotent stem cell of murine teratocarcinoma. When treated with retinoic acid, P19 cells can be differentiated along a neural cell lineage in culture. To isolate peptides that bind to the stem cell, we employed a phage display technology with undifferentiated P19 cells as the target. To reduce nonspecific binding of phages to the cell surface, the phage libraries were preadsorbed to the differentiated P19 cells before each selection on the undifferentiated P19 cells. After eight rounds of the selection, No. 28 phage displaying ALPSTSSQMPQL-peptide was isolated. Immunofluorescence analysis revealed that No. 28 phage selectively binds to the undifferentiated P19 cells but not to the differentiated P19 cells or SHSY cell line. The chemically synthesized peptide ALPSTSSQMPQL presented on the No. 28 phage efficiently inhibited the binding of No. 28 phage to the undifferentiated P19 cells. This result confirmed that No. 28 phage binding to the cell was mediated by the displayed peptide. The identified peptide may be targeted to a marker expressed on the stem cell and thus become a practical tool for the isolation of somatic stem cells.
P19细胞是小鼠畸胎瘤的多能干细胞。用视黄酸处理时，P19细胞在培养中可沿神经细胞谱系分化。为了分离与干细胞结合的肽，我们采用噬菌体展示技术，以未分化的P19细胞为靶点。为减少噬菌体与细胞表面的非特异性结合，在每次以未分化的P19细胞为靶点进行筛选之前，将噬菌体文库预先吸附到分化的P19细胞上。经过八轮筛选，分离出展示ALPSTSSQMPQL肽的28号噬菌体。免疫荧光分析显示，28号噬菌体选择性地与未分化的P19细胞结合，而不与分化的P19细胞或SHSY细胞系结合。28号噬菌体上展示的化学合成肽ALPSTSSQMPQL有效抑制了28号噬菌体与未分化的P19细胞的结合。这一结果证实，28号噬菌体与细胞的结合是由展示的肽介导的。所鉴定的肽可能靶向干细胞上表达的一种标志物，从而成为分离体干细胞的实用工具。
