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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 6, 2005 as doi:10.1096/fj.04-2098fje. |
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* BodiTech Med Inc., Chuncheon, Korea;
Department of Chemistry, Hallym University, Chuncheon, Korea;
Proteogen Inc., Chuncheon, Korea;
Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut, USA; and
|| Department of Genetic Engineering, Hallym University, Chuncheon, Korea
1Correspondence: Department of Genetic Engineering, Hallym University, Chuncheon, Korea 200-702. E-mail: euichoi{at}hallym.ac.kr
SPECIFIC AIMS
We characterized the properties of the Calixarene derivatives as biolinker molecules in immobilization of proteins, compared the sensitivity and the specificity of Calixcrown with those of five other protein attachment agents, and applied Calixcrown chip to analyze samples found in clinical practices.
PRINCIPAL FINDINGS
1. Calixcrown chip shows a superior sensitivity and a much lower detection limit than those chips prepared by other methods
We employed a sandwich immunoassay for detection of specific interaction between prostate specific antigen (PSA) and its antibody pairs as a model system. When we compared the sensitivity of Calixarene derivative with those of four other protein attachment agents, a Calixcrown chip A showed a superior sensitivity and a much lower detection limit than those of other protein chips (Fig. 1
A). The detection limit of Calixcrown chip A was between 100 pg/mL, and the Superaldehyde and the Carboxyl chip were next, but were 10- to 100-fold less sensitive. The biotin-avidin and Protein A chip were the least sensitive with a high detection limit (Fig. 1A, B
). We used an enzyme-substrate interaction for color visualization of on-site deposition of end products on the chips as a more direct method for comparison of sensitivity. The enzyme activity of alkaline phosphatase on the Calixcrown slide was 
10-fold higher than that on the Superaldehyde slide (Fig. 1C
). One possibility of Calixcrown chip’s superior sensitivity is that the proteins immobilized on Calixcrown chip may maintain more intact configuration after their noncovalent attachments to Calixcrown. Other explainations could be that more IgG molecules are bound to the Calixcrown chip or IgG molecules are posed with better exposure pattern to their partners on Calixcrown chip than on other chips.
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2. Calixcrown interacts with protonated amine groups of protein
When fluorescently labeled CRP was immobilized under various ion concentrations of Na+, NH4+, and K+, the presence of only 200 mM NH4+ ion resulted in a complete inhibition of protein immobilization. To the contrary, even 800 mM of K+ ionic concentration was not able to inhibit protein immobilization completely, but only partially, and Na+ ion showed no effect at all as expected from the molecular size of Na+ and crown moiety. This result demonstrated that the protonated amine of protein is an important factor in the interaction with Calixcrown linker. To confirm it, we constructed two mutant Gal4 clones and compared their binding efficiency to Calixcrown molecular linker. When fluorescently labeled oligonucleotide sequence in responding to Gal4 DNA binding domain was probed to detect immobilized two mutant proteins, the fluorescence signal from 9R-Gal4 protein (six His-tags plus nine arginines and Gal4 DNA binding domain) was almost 10 times stronger than that from control Gal4 protein (six His-tags and Gal4 DNA binding domain). This result suggested that extra stretch of basic amino acids of 9R-Gal4 protein contributed to high-density immobilization on Calixcrown monolayer and thus more Cy5-labeled oligonucleotide would bind to 9R-Gal4 protein. Considering most of the –NH2 groups of protein exist as a –NH3+ form in a physiological condition (pH 7.4), this result indicated that Calixcrown interacts with the ammonium ion of protein by ionic interaction.
3. Calixcrown appears to immobilize IgG molecule with right orientation
To show that Calixcrown captures antibody molecule (IgG) with the right orientation, we used the characteristic of Protein A that specifically recognizes the Fc domain of IgG as shown in the schematic diagram of Fig. 2
A. We first immobilized Cy5-labeled IgG molecules on Calixcrown chip A surface and on Superaldehyde glass (Fig. 2B
). We then overlaid Cy3-labeled Protein A on the IgG-immobilized chips. Thus, the Cy5 signal shows the amount of IgG captured on the Calixcrown chip and the Cy3 signal reflects the amount of protein A bound to the Fc region of the immobilized IgG molecule. When the IgG layer was compared, the Calixarene chip showed higher overall Cy5 fluorescence intensity (Fig. 2Ba, b
). It suggests that Calixcrown chip captured or immobilized the IgG molecules more efficiently than the Superaldehyde slide did at the same concentration of the IgG solution. Next, we compared the Cy3 signal to measure the amount of protein A bound to the IgG layer (Fig. 2Bc, d
). Little signal was detected at 4 µg/mL or higher concentration from both chips. One plausible explanation for this would be that at the high concentration each chip was densely packed with the IgG molecules, so that the protein A could not penetrate into the layer. In the meantime, at low concentration, the IgG layer was loosely packed and thus contained some empty room for the access of protein A. Actually, we observed that at 0.16 and 0.8 µg/mL, the fluorescence intensity of the Superaldehyde chip was higher than that of the Calixcrown chip, indicating that more protein A molecules were bound to the IgG layer (Fig. 2Bc, d
). This observation could support our assumption that IgG molecules on the Calixcrown chip may be arranged more regularly with a vertical orientation than IgG molecules on the Superaldehyde slide.
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4. Calixcrown chip can apply to clinical samples and for assay of cellular function
Capture anti-PSA-mAb was immobilized on the Calixcrown chip A and the Superaldehyde chip before serum from a prostate cancer patient was applied. Subsequently, when Cy5-labeled detector antibody of the sandwich pair was probed to detect antigens in serum, the fluorescence intensity on Calixcrown chip A was as much as 100-fold higher than that of Superaldehyde chip. For detection of anti-gp120 antibody against HIV virus in serum, Cy5-labeled anti-human-IgG and HIV gp120 recombinant protein were used as a detector and a capture molecule, respectively. Strong signal was detected on the Calixcrown chip even after 105-fold dilution of the HIV serum, whereas the Superaldehyde chip gave a weak signal at 104-fold dilution. We applied the Calixcrown chip A to identify up- or down-regulated proteins using antibody microarray against 36 varieties of cellular proteins. Compared with normal rat liver, we were able to detect 7 up-regulated proteins including caspase-3 and integrin, and 3 down-regulated proteins from Dimethylnitrosamine-treated rat liver.
CONCLUSIONS AND SIGNIFICANCE
By comparing other immobilization methods in this study, we found that Calixcrown-coated slide showed a higher efficiency of detection while maintaining a low background level (Fig. 1)
. The superiority of the Calixarene chip over the Superaldehyde slide could be explained by the observation that protein-immobilization via ionic interaction may maintain a better folding conformation and keeps the protein more functional, although the electrostatic charges present on the glass may lead to the denaturation of the proteins. The directional immobilization of proteins on a solid surface is one of the most powerful advantages of the Calixcrown molecular linker.
The merits of the oriented immobilization of proteins are good steric accessibility of the active binding site and the increased stability. The Protein A binding experiment suggests more oriented immobilization of antibodies on Calixcrown slide than on Superaldehyde slide (Fig. 2)
. Since the amine group is the primary guest moiety for complex formation with host Calixcrown molecule, introduction of extra positive amino acids into a protein helps a more efficient site-specific attachment of proteins to the solid surface. Thus, in case Calixcrown is used as a molecular linker agent, inserting a stretch of basic amino acids such as arginine and lysine into a protein is expected to become an easy and convenient method for the oriented immobilization of protein on a solid surface.
Our study showed that the Calixcrown chip can be used as a biological tool with a wide range of applications, including protein-protein interaction, protein-DNA interaction, and an enzyme activity assay. The Calixarene derivative as a fabrication agent for protein immobilization by noncovalent ionic interaction provides several advantages (Fig. 3
). First, the binding reaction between Calixcrown and protein occurs spontaneously without any energy input so that protein is immobilized more easily than in other methods. Second, changes of affinity or specificity in the protein–protein interaction can be minimized since there is no chemical reaction involved during the protein immobilization process. Third, high-density immobilization can be achieved by guided interaction between guest antibody and host Calixcrown bi-functional linker.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2098fje;
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