Ni-NTA or Cu-NTA sufaces

Application Note:

A protein molecule is linked to the surface only through the poly-His tag with controlled orientation but otherwise stays away from the surface as much as possible. This ensures protein activity of the native state. No sample pre-purification is needed.

Figure 1. The zero background Cu2+ surface consists of chelated Cu2+ ion tethered to the high-density PEG coating. A protein molecule is attached specifically via a poly-His tag.

Recombinant proteins are often produced with poly-histidine (His) tags to facilitate purification. These proteins are now used in the fabrication of protein microarrays. To take advantage of the availability of the poly-His tag, we have developed a chelated Cu2+ surface based on the zero background PEG coating, as shown in Figure 1. The use of this surface essentially eliminates the purification step and incorporates it directly in the protein immobilization process, i.e., one can directly use the crude lysate in microarray fabrication, as illustrated in Figure 2 for green fluorescent protein (GFP). 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. Both the inertness of the chemical surrounding and the controlled orientation contribute to an ideal environment for the immobilized protein molecule to retain its native conformation and activity, as illustrated in Figure 3. For 6xHis tagged sulfotransferase and alkaline phosphatase immobilized with controlled orientation on the Cu2+/PEG surface, their activities are nearly identical to those in the solution phase. For comparison, enzymes immobilized with random orientation on the 3-aminopropyltriethoxysilane (gamma-APS) coated surface show only ~10% of the activities.


Figure 3. A comparison of enzyme activities in the solution phase, with those of random orientation on gamma-APS surface or controlled orientation on the Cu2+/PEG surface.	Figure 2. The left shows the intrinsic fluorescence of 6xHis tagged GFP adsorbed on the chelated Cu2+/PEG surface. The surface resists the non-specific adsorption of GFP without His tag (right). Spot diameter ~0.2 mm.

The repulsive 2D chemical environment on the Cu2+/PEG surface is of critical importance not only to the activity of immobilized protein molecules, but also to the maintenance and revival of this activity. The latter is demonstrated in fluorescence microscope images (Figure 4) obtained after repeated cycles of denaturing and refolding of 6xHis-GFP immobilized on three different surfaces: (a) the Cu2+/PEG surface; (b) Cu2+ ions chelated to surface iminodiacetic acid groups on a 3-aminopropyltriethoxysilane (gama-APS) functionalized surface; and (c) the gamma-APS surface for nonspecific protein adsorption. The intrinsic fluorescence intensities are very different on the three surfaces, although immunostaining reveals similar amounts of GFP. The fluorescence intensity on the Cu2+ /gamma-APS or the gamma-APS surface is 70% or < 20% of that on the Cu2+/ PEG surface. While no fluorescence is detected after the denaturing step on all three surfaces, result of refolding is a strong function of the chemical nature of the surface. On the Cu2+/PEG surface, most of the fluorescence intensity is recovered after the refolding step. In contrast, little fluorescence intensity is left on the Cu2+/gamma-APS or gamma-APS surface. On the Cu2+/PEG surface, each immobilized GFP molecule is linked only by the 6xHis tag but otherwise prefers to stay away from the surface due to the repulsive nature of the PEG functionality. This repulsive or non-fouling nature of the surface ensures that the weak protein-surface interaction does not introduce additional barriers on the energy landscape for protein refolding. On the gamma-APS surface, there is attractive and non-specific interaction between GFP and the “sticky” or fouling –NH2 functional groups in the immediate surrounding. Upon denaturing, such non-specific interaction with the sticky environment is expected to increase, thus effectively introduces insurmountable barriers on the energy landscape for protein refolding. The capability of the Cu2+/PEG surface for on-chip refolding of protein molecules opens the door to many potential applications, e.g., direct on-chip generation of protein microarrays, the removal of inclusion body from recombinant proteins, etc. These coatings are available on standard microscope slides, cover slips, & silicon wafers. Our customers have successfully applied the Cu2+/PEG surfaces for a range of applications, including protein sensors, protein microarrays, single molecule spectroscopy, biological atomic force microscopy and other biophysical studies. Coming soon: a three-dimensional (3D) version of the Cu2+/PEG surface is under development. The large surface area afforded by the microporous 3D coating allows high loading of poly-His tagged probe molecules. This is ideal for the detection of very low concentrations of biomarkers.

Figure 4. Fluorescence microscope images of 6xHis-GFP before and after cycles of denaturing (De) and refolding (Re) on three surfaces: (a) the Cu2+/PEG; (b) Cu2/ /gamma-APS; (c) gamma-APS. Denaturing involves immersion of the 6xHis-GFP coated surface in a buffer solution at pH = 3.5, while refolding corresponds to incubating the sample with a buffer solution containing 1xPBS, 20% sucrose and 10% glycerol at pH = 8.1.

READ Publications using our Cu2+ surfaces: Proteomics, 2005, 5, 416; Proteomics, 2007, 7, 1771; Nature Method, 2008, 5, 507; BMC Biotech., 2009, 1. etc..