The traditional methods of producing protein arrays require the separate in vivo expression of hundreds or thousands of proteins, followed by separate purification and immobilization of the proteins on a solid surface. Cell-free protein array technology attempts to simplify protein microarray construction by bypassing the need to express the proteins in bacteria cells and the subsequent need to purify them. It takes advantage of available cell-free protein synthesis technology which has demonstrated that protein synthesis can occur without an intact cell as long as cell extracts containing the DNA template, transcription and translation raw materials and machinery are provided. Common sources of cell extracts used in cell-free protein array technology include wheat germ, Escherichia coli, and rabbit reticulocyte. Cell extracts from other sources such as hyperthermophiles, hybridomas, Xenopus oocytes, insect, mammalian and human cells have also been used.
The target proteins are synthesized in situ on the protein microarray, directly from the DNA template, thus skipping many of the steps in traditional protein microarray production and their accompanying technical limitations. More importantly, the expression of the proteins can be done in parallel, meaning all the proteins can be expressed together in a single reaction. This ability to multiplex protein expression is a major time-saver in the production process.
Various research groups have developed their own methods, each differing in their approach, but can be summarized into 3 main groups. Nucleic acid programmable protein array (NAPPA): NAPPA uses DNA template that has already been immobilized onto the same protein capture surface. The DNA template is biotinylated and is bound to avidin that is pre-coated onto the protein capture surface. Newly synthesized proteins which are tagged with GST are then immobilized next to the template DNA by binding to the adjacent polyclonal anti-GST capture antibody that is also pre-coated onto the capture surface (Figure 1). The main drawback of this method is the extra and tedious preparation steps at the beginning of the process: (1) the cloning of cDNAs in an expression-ready vector; and (2) the need to biotinylate the plasmid DNA but not to interfere with transcription. Moreover, the resulting protein array is not ‘pure’ because the proteins are co-localized with their DNA templates and capture antibodies.
Protein in situ array (PISA): Unlike NAPPA, PISA completely bypasses DNA immobilization as the DNA template is added as a free molecule in the reaction mixture. In 2006, another group refined and miniaturized this method by using multiple spotting technique to spot the DNA template and cell-free transcription and translation mixture on a high-density protein microarray with up to 13,000 spots (Figure 2). This was made possible by the automated system used to accurately and sequentially supply the reagents for the transcription/translation reaction occurs in a small, sub-nanolitre droplet.
In situ puromycin-capture: This method is an adaptation of mRNA display technology. PCR DNA is first transcribed to mRNA, and a single-stranded DNA oligonucleotide modified with biotin and puromycin on each end is then hybridized to the 3’-end of the mRNA. The mRNAs are then arrayed on a slide and immobilized by the binding of biotin to streptavidin that is pre-coated on the slide. Cell extract is then dispensed on the slide for in situ translation to take place. When the ribosome reaches the hybridized oligonucleotide, it stalls and incorporates the puromycin molecule to the nascent polypeptide chain, thereby attaching the newly synthesized protein to the microarray via the DNA oligonucleotide (Figure 3). A pure protein array is obtained after the mRNA is digested with RNase. The protein spots generated by this method are very sharply defined and can be produced at a high density.
Nano-well array formats are used to express individual proteins in small volume reaction vessels or nano-wells (Figure 4). This format is sometimes preferred because it avoids the need to immobilize the target protein which might result in the potential loss of protein activity. The miniaturization of the array also conserves solution and precious compounds that might be used in screening assays. Moreover, the structural properties of individual wells help to prevent cross-contamination among chambers.
DNA array to protein array (DAPA) is a method developed in 2007 to repeatedly produce protein arrays by ‘printing’ them from a single DNA template array, on demand (Figure 5). It starts with the spotting and immobilization of an array of DNA templates onto a glass slide. The slide is then assembled face-to-face with a second slide pre-coated with a protein-capturing reagent, and a membrane soaked with cell extract is placed between the two slides for transcription and translation to take place. The newly-synthesized his-tagged proteins are then immobilized onto the slide to form the array. Over 20 protein arrays can be printed from a single DNA array with no adverse effects on production efficiency.