Site-Specific Immobilization of Proteins in a Microarray

Abstract

The completion of the Human Genome Project has generated enormous opportunities, as well as challenges, to protein scientists. The key issue now is to develop efficient strategies that allow high-throughput studies of many thousands of new proteins. The DNA microarray technology makes it possible for simultaneous expression profiling of thousands of genes from various biological sources. However, it is now well known that, at the cellular level, the relative abundance of messenger RNAs does not always correlate to their protein expression level (1). Therefore, it is essential to study the large number of proteins present in an organism in order to better understand its molecular functions. Protein microarray, which adopts the same spotting technology used to fabricate DNA microarray, has recently been developed (2–5). It promises to provide a means for high-throughput identification and quantification of proteins from different biological samples. In a protein microarray, tens of thousands of proteins may be immobilized on a solid surface, such as a glass slide (4,5), and the screening of protein activities could be carried out simultaneously. One of the main challenges in the fabrication of protein microarrays currently is the ability to immobilize proteins in their native conformation on surfaces, while preserving their active sites for functional studies (5,6). Several approaches have been developed (for review, see ref. 2). In most cases, however, these modes of protein attachment are unspecific, causing the molecules to be immobilized in the <wrong” orientation. A number of strategies have recently been reported, which allow site-specific immobilization of molecules in the microarray format (5–8). So far, there has been only one report of site-specific attachment of proteins on glass slides using His-tag (5). However, the binding between Ni-NTA and His-tag proteins is not very stable, and is often susceptible to interference by many commonly used chemicals and salts (9). A couple of new methods have recently been proposed that allow site-specific covalent immobilization on surfaces, but they remain to be experimentally demonstrated on the glass slide used in a microarray (10,11).