Niro Ramachandran, Ph.D.

Novel Approaches to Protein Arrays

Existing protein arrays involve the tedious and lengthy process of expressing proteins in living cells followed by purifying, stabilizing, and spotting the samples. This process is a bottleneck in the preparation of the arrays. Moreover, functionally active proteins require careful manipulation, and the less that is needed the better. Our approach to developing a protein array, a Nucleic Acid-Programmable Protein Array (NAPPA), replaces the complex process of spotting purified proteins with the straightforward and much simpler process of spotting plasmid DNA. All genes are then simultaneously transcribed/translated in a cell-free system and the resulting proteins are immobilized in situ, minimizing direct manipulation of the proteins and making this approach well suited to high-throughput applications.

Figure 1 Schematic representation of screening protein-protein interactions with NAPPA. (A) On each spot, a target plasmid and an affinity capture molecule are linked to the prepared glass slide. (B) The slide is bathed with the cell-free in vitro transcription/translation mix, containing one or more query plasmids expressing different affinity tags. (C) The target proteins are expressed and immobilized on the spots, as are query proteins if they bind to that target. (D) The wells are then washed to remove unbound protein. (E) Target-query complexes are detected by fluorescence.  Enlarge

NAPPA in progress...

Step 1
Expression and Immobilization of Target Proteins

We were able to reliably express and specifically immobilize multiple in vitro synthesized targets on to α-GST coated plate. As shown in figure 2, different constructs were expressed in each row, and subsequent detection was carried out by treating each column with a protein specific antibody to identify target proteins.

Figure 2 In situ expression and Immobilization of target proteins.  Enlarge

Step 2
Protein-Protein Interaction

Here we detect the interaction between transcription factors Jun and Fos. Fos-GST was expressed, immobilized on α-GST plate and detected by Fos specific antibody confirming protein expression. Fos-GST was also co-expressed with HA-Jun and their interaction was detected by both Jun specific and HA epitope specific antibody. The Jun-Fos interaction was detected clearly above background signal (Figure 3). Other interactions investigated among p16 and CDKs showed specific interactions among and CDK4-p16 and CDK6-p16, whereas no interaction was observed between CDK2 and p16, in accordance with published data.

Figure 3 a) Protein-Protein interaction between transcription factors Jun and Fos. Fos-GST is co-expressed with HA-Jun, and their interaction is detected by Jun specific antibody and HA epitope specific antibody.   Enlarge

b) Detection with α-HA antibody shows sensitivity of detection of the interaction   Enlarge

Step 3
NAPPA based Microarray

NAPPA has been scaled down to microarray format on glass slides. First, GST antibody and cDNAs are linked to aminosilane treated glass slides. The cDNA array is then bathed in rabbit reticulocyte lysate to express target proteins. Target proteins are expressed as C-terminus GST fusions and as such are immobilized onto GST specific antibody (pAB). Detection of all expressed proteins is carried out with GST specific antibody (mAB).

Figure 4 NAPPA Microarray. Genes (35) involved in eukaryotic DNA replication are expressed and immobilized in situ as GST fusion proteins on a microarray format. Expression of target proteins was detected by fluorescence. The samples were arrayed using a GMS 417 pin arrayer with 900µm spacing.  Enlarge