Recent Research Developments

Index of Recent Research News
March 3rd, 2004
    Enzymatic activity on a chip: the critical role of protein orientation

    One of the most exciting tools in proteomics is the protein microarray technology, in which a large number of proteins or peptides are immobilized on a solid substrate for the high-throughput, parallel analysis of biochemical properties and biological activities. Compared to its counter part in genomics (i.e., DNA microarrays), there are two inherent difficulties associated with protein immobilization: i) Background. Proteins tend to adsorb nonspecifically to solid substrates, leading to not only the possibility of denaturation but also background problems during assays; ii) Conformation and orientation. Because proteins have complex structures and activities, the immobilization chemistry has to be such that it preserves a protein in native state and with optimal orientation for protein-target interaction.

    Figure 1. Schematic illustration of the approach for forming oriented protein molecules on the surface.

    Recent research by graduate student Taewoon Cha, chemistry professor Xiaoyang Zhu, and their collaborator, Dr. Athena Guo of MicroSurfaces, Inc., has demonstrated an elegant strategy for oriented protein microarrays using recombinant poly-histidine tags and surface chelated metal ions on an otherwise "zero" background surface. There are a number of advantages associated with this strategy for the fabrication of protein microarrays. The generation of a poly-His tag to either the C-terminus or N-terminus is perhaps the most commonly used method in recombinant protein technology. Unlike other fusion protein strategies, the poly-His tag approach for purification can be applied not only to proteins in native states, but also to those under denature conditions or to small peptides. When applied to protein microarray technology, this strategy effectively combines the steps of purification and immobilization. To take advantage of the high specificity of binding between a poly-His tag and chelated metal ions, the surface must resist the non-specific adsorption of all other protein molecules lacking the poly-His tag. For this purpose, these researchers used high density poly(ethylene glycol) (PEG) films on silicon surfaces. The intrinsic inertness of the PEG functionality permits minimal non-specific adsorption of proteins, while the readily available alcohol functional groups on the surface of the PEG film can be easily activated for metal ion adsorption. Except for the poly-His tag on the N- or C-terminus, each immobilized protein molecule stays away from and minimizes its interaction with the surface due to the repulsive nature of the PEG environment. As a result, there is minimal disturbance to the native conformation of the protein. The authors used a model protein, green fluorescent protein (GFP), and an enzyme, sulfotransferase (STa-IV) in the study. A paper on this subject is coming out in the biotech journal Proteomics.

    Figure 2 Fluorescence microscope images of: (A) 6xHis-GFP and (B) GFP on the Cu2+-IDA-mPEG-Si surface (C) purified 6xHis-STa-IV on the Cu2+-IDA-mPEG-Si surface; and (D) background test which involves spotting 6xHis-STa onto a DEIDA-mPEG-Si surface after pre-treatment with CuSO4 solution. (E) crude lysate containing 6xHis-STa-IV on the Cu2+-IDA-mPEG-Si surface; and (F) background test which involves spotting of crude lysate containing 6xHis-STa-IV onto the DEIDA-mPEG-Si surface after pre-treatment with CuSO4 solution.

    Most recently, these researchers have also established the critical importance of controlling the orientation of immobilized molecules in enzyme kinetics. While oriented STa selectively immobilized on the PEG surface via the 6xHis tag faithfully reflect activities of solution phase proteins, those with random orientation on the surface do not. This observation can be understood because the active sites on certain population of randomly oriented protein molecules on the surface are not accessible. The possible presence of multiple covalent bonds between a randomly oriented protein molecule and the solid surface may also affect its conformation. These authors concluded that controlling the orientation of immobilized protein molecules and designing an ideal local chemical environment on the solid surface are both essential if protein microarrays are to be used as quantitative tools in biomedical research. This exciting finding has also been communicated to Proteomics. This collaborative project has been funded by NIH and NSF.

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