迄今为止，蛋白阵列（protein arrays）主要有两种形式：一种是抗体阵列（antibody arrays），利用阵列上的抗体识别样品中的蛋白或其他分子；另一种则是我们今天要谈到的靶蛋白阵列（target protein arrays），通过阵列上的蛋白检测这些我们感兴趣的蛋白和其他分子，如药物、抗体、核酸、脂类或其他蛋白的作用。由于有高通量的优点，蛋白阵列是研究蛋白功能的好工具。但是，通常的靶蛋白阵列生产方式是分别得到不同的蛋白，纯化后再借助各种化学连接将它们点样到阵列上。这样的方式面临面临两个难题：一个是如何高通量的产生和纯化各种蛋白；另一个是如何保持蛋白在点样后的稳定性。虽然这种方式已经证明可行，不过这些技术上的难题却导致其难以广泛地被采用。
最新的一期《Science》（July 2，2004）报道了Harvard Medical School的科学家制作蛋白芯片的新方法，能够使其更实用：研究者将编码经过抗原决定簇标记的靶蛋白（epitope-tagged target proteins）的cDNA印记在玻璃载片上，然后利用无细胞的系统表达蛋白，再利用抗原决定簇标记进行原位固定。这种方法既不需要对不同的蛋白分别进行表达和纯化，又可以在使用前才产生蛋白，从而解决了保持蛋白稳定的难题。具体说，为了既能够附着DNA，又能够保持DNA的构向以利于转录和翻译，研究者利用紫外线将补骨脂素－生物素（psoralen-biotin）和DNA表达质粒接合在一起。而且，每一个被表达的蛋白的碳端都标记了一个谷胱甘肽S- 转移酶(Glutathione S-transferase，GST)，能够通过和预先印记在表达质粒旁边的GST抗体作用，从而使表达的蛋白固定在阵列上。（如下图）
那这种核酸可编程性蛋白阵列（nucleic acid programmable protein array, NAPPA）到底能不能满足高通量的检测要求呢？研究者利用这项新技术检测了29个人类DNA复制起始蛋白的两两互作。其中，特别研究了复制许可因子Cdt1和特定蛋白的结合的调控。甚至还对Cdt1和另一蛋白Geminin结合所需的结构域进行了精细的作图。
不过像其他的高通量的研究手段，NAPPA也有美中不足：首先，体外转录翻译系统中可能存在一些桥梁蛋白和抑制因子可能影响结果的准确；其次，多肽标记（peptide tag）的使用，可能会因为空间效应影响蛋白的结合区而使蛋白无法结合；最后，NAPPA的蛋白体外表达系统缺乏后翻译的修饰（posttranslational modification），也会因为无法满足体内蛋白表达的时间和空间的分隔，导致蛋白的折叠和活性的变化。无论如何，NAPPA的出现，给生产蛋白芯片提供了更多的途径，是一个令人鼓舞的重大突破。
Self-Assembling Protein Microarrays
Protein microarrays provide a powerful tool for the study of protein function. However, they are not widely used, in part because of the challenges in producing proteins to spot on the arrays. We generated protein microarrays by printing complementary DNAs onto glass slides and then translating target proteins with mammalian reticulocyte lysate. Epitope tags fused to the proteins allowed them to be immobilized in situ. This obviated the need to purify proteins, avoided protein stability problems during storage, and captured sufficient protein for functional studies. We used the technology to map pairwise interactions among 29 human DNA replication initiation proteins, recapitulate the regulation of Cdt1 binding to select replication proteins, and map its geminin-binding domain.
Fig. 1. NAPPA approach. Biotinylation of DNA: Plasmid DNA is cross-linked to a psoralen-biotin conjugate with the use of ultraviolet light (17). (A) Printing the array. Avidin (1.5 mg/ml, Cortex), polyclonal GST antibody (50 µg/ml, Amersham), and Bis (sulfosuccinimidyl) suberate (2 mM, Pierce) are added to the biotinylated plasmid DNA. Samples are arrayed onto glass slides treated with 2% 3-aminopropyltriethoxysilane (Pierce) and 2 mM dimethyl suberimidate. 2HCl
Fig. 2. Expression of target proteins and detection of protein interactions on a NAPPA microarray format. (A) Eight target plasmid DNAs encoding C-terminal GST fusion proteins in pANT7_cGST (fig. S2) were immobilized onto the glass slide at a density of 512 spots per slide (900-µm spacing). The target proteins were expressed with 100 µl rabbit reticulocyte lysate supplemented with T7 polymerase. Signals were detected with antibody to GST and tyramide signal amplification (TSA) reagent (PerkinElmer). To verify that the detected proteins were the expected target proteins, and to confirm that there was no cross-talk across the slide, we used target protein–specific antibodies, which detected only their relevant spots (fig. S3). (B and C) The eight genes were queried for potential interactors with (B) Jun and (C) p16. Query DNA encoding an N-terminal HA tag was added to the reticulocyte lysate before expressing the target proteins (fig. S2). Target and query proteins were coexpressed and the interaction was detected with an antibody to HA (12CA5). The bar graphs show average intensity (+SD) from 64 samples for each interaction. Images were quantified using ScanAlyze software (Michael Eisen, Lawrence Berkeley National Laboratory, CA). The signals were corrected for local background.
Fig. 4. Characterization of Cdt1. (A) Cdt1 regulation. NAPPA was used to test whether Cdc6 could act as a bridging protein between Cdt1 and MCM2. Target proteins Cdt1, Cdc45 (negative control), and MCM5 (positive control) were expressed in duplicate (top panel) and confirmed by an antibody to GST. The target proteins were probed with either HA-MCM2 alone (left panel) or in the presence of coexpressed His-Cdc6 (right panel). The binding of MCM2 was detected with an antibody to HA. (B) Cdt1 deletion mapping. NAPPA was used to map the binding domain of geminin on Cdt1. Fragments from various regions of Cdt1 (as indicated) were generated by PCR and cloned into target expression vectors. The partial or full-length polypeptides were expressed and detected on the array with an antibody to GST (left panel). To identify the binding region of geminin, the array was queried with HA-geminin (right panel) and developed with an antibody to HA. Vertical lines (dashed) delimit the small (15-aa) region common to all the fragments that bound geminin. A small 78-aa domain (135 to 212 aa) containing the noted 15 aa (198 to 212 aa) was expressed along with full-length Cdt1 (bottom left), which was again queried with geminin (bottom right).
