Generation of High Density Protein Microarrays by Cell-free in Situ Expression of Unpurified PCR Products

Abstract

Due to the success of DNA microarrays and the growing numbers of available protein expression clones, protein microarrays have become more and more popular for the high throughput screening of protein interactions. However, the widespread applicability of protein microarrays is currently hampered by the large effort associated with their production. Apart from the requirement for a protein expression library, expression and purification of the proteins themselves and the lacking stability of many proteins remain the bottleneck. Here we present an approach that allows the generation of high density protein microarrays from unbound DNA template molecules on the chip. It is based on the multiple spotting technique and comprises the deposition of a DNA template in a first spotting step and the transfer of a cell-free transcription and translation mixture on top of the same spot in a second spotting step. Using wild-type green fluorescent protein as a model protein, we demonstrated the time and template dependence of this coupled transcription and translation and showed that enough protein was produced to yield signals that were comparable to 300 g/ml spotted protein. Plasmids as well as unpurified PCR products can be used as templates, and as little as 35 fg of PCR product (ϳ22,500 molecules) were sufficient for the detectable expression of full-length wild-type green fluorescent protein in subnanoliter volumes. We showed that both aminopropyltrimethoxysilane and nickel chelate surfaces can be used for capture of the newly synthesized proteins. Surprisingly we observed that nickel chelate-coated slides were binding the newly synthesized proteins in an unspecific manner. Finally we adapted the system to the high throughput expression of libraries by designing a single primer pair for the introduction of the required T7 promoter and demonstrated the in situ expression using 384 randomly chosen clones. Molecular & Cellular Proteomics 5:1658 -1666, 2006.The understanding of complex cellular networks necessitates tools that are amenable to the analysis of different parameters in a highly parallel manner (1). Although in the last years DNA microarrays were the technology of choice to monitor the abundance of several thousands of mRNA transcripts at a time, such studies provide us with little information on the proteins that are encoded by these transcripts (2, 3). However, because proteins rather than DNA carry out cellular functions, there is large interest to analyze proteins and their entirety, the proteome, in a manner comparable to DNA microarrays. One technology that is envisaged to meet the demands of high throughput protein interaction and modification screening is protein microarray technology (4 -8).Protein microarrays have been applied in different areas of application, such as the analysis of protein-protein interactions (9 -11), the identification of substrates for protein kinases (12-14), or the elucidation of potential diagnostic markers in bacterial or autoimmune diseases (15-18). All of them share the bas...

Precision proteomics: The case for high resolution and high mass accuracy

“…However, the challenge associated with expressing thousands of full-length proteins and immobilizing them in a native state on a chip is daunting (5,6). In practice, the role of protein arrays has been limited, and the literature contains few examples of their successful use.…”

Next generation microfluidic platforms for high-throughput protein biochemistry

“…The use of plasmids as the template is suboptimal as plasmid generation also requires long and tedious cloning and transformation steps. Angenendt et al have shown that PCR products can serve directly as templates for protein synthesis and protein array production, further streamlining the approach [11]. A second problem associated with the NAPPA method is that synthesis is not compartmentalized.…”

Large-Scale Protein-Protein Interaction Detection Approaches: Past, Present and Future

“…The binding of Hisx6-tagged green fl uorescence protein (GFP) and untagged GFP to Ni surface was compared by probing immobilized proteins with anti-GFP and anti-penta-His antibodies. Anti-GFP antibodies produced similar signals while anti-penta-His antibody signal was seen only in Hisx6-GFP, suggesting that unspecifi c binding to Ni slides rather than uniform attachment of Hisx6 tag to Ni may be responsible for protein binding (1). Other limitations of utilizing Ni-His affi nity in functional microarrays arise from the fact that Ni-His interaction can be disrupted by washing, prolonged storage or commonly used reagents including EDTA and DTT (57).…”

“…Interestingly, the assumption that the binding of Hisx6-labeled proteins to Ni chips is dependent on His tag has been recently challenged (1). The binding of Hisx6-tagged green fl uorescence protein (GFP) and untagged GFP to Ni surface was compared by probing immobilized proteins with anti-GFP and anti-penta-His antibodies.…”

“…GST-fused proteins attach directly to glutathione-coated slide, bypassing the need for protein purifi cation and storage. However, NAPPA requires cDNA cloning, plasmid immobilization and yields low density microarrays (1). A more advanced cell-free expression approach allows protein production from even unpurifi ed PCR products and consists of spotting of DNA template followed by transcription and translation mix deposition on a microchip with subsequent spot rehydration to initiate separate expression reactors (1).…”