Faix Peggy Ho, Burg Michael A, Gonzales Michelle, Ravey Edward P, Baird Andrew, Larocca David
Selective Genetics, Inc., San Diego, CA, USA.
Biotechniques. 2004 Jun;36(6):1018-22, 1024, 1026-9. doi: 10.2144/04366RR03.
Phage display technologies are powerful tools for selecting binding ligands against purified molecular targets, live cells, and organ vasculature. However, the selection of natural ligands using phage display has been limited because of significant problems associated with the display of complex cDNA repertoires. Here we describe the use of cDNA fragmentation and open reading frame (ORF) selection to display a human placental cDNA library on the pIII coat protein of filamentous phage. The library was enriched for ORFs by selecting cDNA-beta-lactamase fusion proteins on ampicillin, resulting in a cDNA population having 97% ORFs. The ORF-selected cDNAs were fused to pIII in the phagemid vector, pUCMG4CT-198, and the library was rescued with a pIII-deleted helper phage for multivalent display. The resulting phagemid particle library consisted of 87% ORFs, compared to only 6% ORFs when prepared without ORF selection. Western blot analysis indicated cDNA-pIII fusion protein expression in eight out of nine ORF clones tested, and seven of the ORF encoded peptides were displayed multivalently. The high level of cDNA expression obtained by ORF selection suggests that ORF-enriched phage cDNA libraries prepared by these methods will be useful as functional genomics tools for identifying natural ligands from various source tissues.
噬菌体展示技术是用于筛选针对纯化分子靶标、活细胞和器官脉管系统的结合配体的强大工具。然而，由于与复杂cDNA文库展示相关的重大问题，利用噬菌体展示筛选天然配体一直受到限制。在此，我们描述了使用cDNA片段化和开放阅读框（ORF）筛选，在丝状噬菌体的pIII外壳蛋白上展示人胎盘cDNA文库。通过在氨苄青霉素上筛选cDNA-β-内酰胺酶融合蛋白，该文库富集了ORF，从而得到一个具有97% ORF的cDNA群体。将经ORF筛选的cDNA与噬菌粒载体pUCMG4CT-198中的pIII融合，并使用缺失pIII的辅助噬菌体拯救该文库以进行多价展示。所得的噬菌粒颗粒文库由87%的ORF组成，而未进行ORF筛选时制备的文库中ORF仅占6%。蛋白质免疫印迹分析表明，在所测试的9个ORF克隆中有8个克隆表达了cDNA-pIII融合蛋白，并且7个ORF编码的肽进行了多价展示。通过ORF筛选获得的高水平cDNA表达表明，通过这些方法制备的富含ORF的噬菌体cDNA文库将作为功能基因组学工具，用于从各种来源组织中鉴定天然配体。
