UPA, a universal protein array system for quantitative detection of protein-protein, protein-DNA, protein-RNA and protein-ligand interactions

Abstract

Protein-protein interactions have been widely used to study gene expression pathways and may be considered as a new approach to drug discovery. Here I report the development of a universal protein array (UPA) system that provides a sensitive, quantitative, multi-purpose, effective and easy technology to determine not only specific protein-protein interactions, but also specific interactions of proteins with DNA, RNA, ligands and other small chemicals. (i) Since purified proteins are used, the results can be easily interpreted. (ii) UPA can be used multiple times for different targets, making it economically affordable for most laboratories, hospitals and biotechnology companies. (iii) Unlike DNA chips or DNA microarrays, no additional instrumentation is required. (iv) Since the UPA uses active proteins (without denaturation and renaturation), it is more sensitive compared with most existing methods. (v) Because the UPA can analyze hundreds (even thousands on a protein microarray) of proteins in a single experiment, it is a very effective method to screen proteins as drug targets in cancer and other human diseases.

Figure 1

Protein–protein interactions monitored with the use of a universal protein array. (A) Autoradiographic signals detected from the array that was incubated with the ³²P-labeled GST-K–p52 and washed with buffer A100. (B) Signals detected after washing the array (from A) with buffer A1000. (C) Quantitative representation of relative affinities (in reading units from a densitometer) of 48 proteins for the transcriptional coactivator p52 after washing with 1000 mM KCl.

Figure 2

Protein–DNA, protein–RNA and protein–ligand interactions monitored with the use of the universal protein array. Autoradiographic signals detected from the same array which was repeatedly stripped and incubated with the ³²P-labeled dsDNA probe (A), the ssDNA probe (B), the RNA probe (C) and the ¹²⁵I-labeled ligand T3 (D) after the array was washed with 500 mM KCl.

Figure 3

Use of UPA to detect ASF/SF2-interacting proteins. (A) Sixteen selected proteins (or fractions) were analyzed for interaction with ³²P-labeled 6H(K)ASF/SF2. CTD, the C-terminal domain of RNA polymerase II fused to GST; RPB5, RPB6, RPB8, RPB10α and RPB10β correspond to individual subunits of RNA polymerase II fused to GST; TBP, TATA-binding protein; f:TFIID, affinity-purified flag-tagged TBP-containing TFIID complex from HeLa cells; RXR, retinoid-X receptor; TR, thyroid hormone receptor; His-H1, histone H1; Co-His, co-histones; HMG1, high mobility group protein 1; ASF, alternative splicing factor; GST–Nu, GST–nucleolin fusion; GST-K, GST fused with a synthetic heart muscle kinase site. (B) Autoradiographic detection of UPA after washing the membrane with 100 mM KCl. (C) Signals detected after washing the membrane with 500 mM KCl.