Next-generation high-density self-assembling functional protein arrays

Abstract

We developed a high-density self-assembling protein microarray, based on the nucleic acid programmable protein array (NAPPA) concept, to display thousands of proteins that are produced and captured in situ from immobilized cDNA templates. We arrayed up to 1,000 unique human cDNAs and obtained high yields of protein expression and capture with minimal variation and good reproducibility. This method will enable various experimental approaches to study protein function in high throughput.

Figure 1

Test array. (a) A test array representing 96 unique genes was printed and stained with PicoGreen dye to show DNA binding (left panel). The negative control includes a non expressing plasmid. Test arrays were expressed using T7 transcription and translationally-coupled rabbit reticulocyte lysate and the proteins were detected using an anti-GST antibody. Two slides were processed on the same day (middle two panels) and a third slide was processed on a different day (right panel). (b)The amount of protein produced in each feature is shown as a log plot. A 99% success was attained for protein signal, based on a cut off of 3 SD above the negative control (features containing a non expressing plasmid). (c) The raw protein signal from arrays processed on the same day and on different days was compared. Average correlation of signal was >0.95.

Figure 2

High density array. (a) A high density array with DNA representing a thousand unique genes was printed in duplicate (see Supplementary Table 1 for gene list). The arrays were stained with PicoGreen to show DNA binding (left panel) and anti-GST antibody after expressing the arrays using cell free expression lysate (right panel). (b) The protein signal at every feature is plotted against the amount of DNA captured there. Most of the DNA (98%) and protein signals (92%) were within 2 fold of their respective means. The background (3 SD above the signal from the negative control) is indicated with a line across the graph. (c) The respective success of protein signal was examined by protein class and size and shown as a log plot.

Figure 3

Protein interactions on high density arrays. (a) An array containing DNA for 647 genes was printed (see Supplementary Table 2 for gene list). DNA binding was confirmed by PicoGreen staining and protein signal was confirmed by staining with an anti-GST antibody. (b) Cell free expression lysate was supplemented with query DNA sub-cloned in pANT7_nHA (100–300 ng) expressing query proteins Jun, Fos and MDM2. The binding of the query to the target proteins on the array was detected using the appropriate protein specific antibodies. A control array was processed in each case where no query plasmid was added to the cell free expression lysate. The graph below shows the trimmed mean signal (25%–75%) for each array and the average of the replicate signals for the target protein.