생물학적 응용을 위한 단백질 마이크로어레이에 관한 연구

박병철, 박성구, 윤갑석, 조사연, 이도희, 김승호

생명공학연구원 단백질공학실

Printing Proteins As Microarray For Biological Applications

B. C. Park, S. G. Park, G.-S. Yoon, S. Y. Cho, D. H. Lee, S.-H. Kim

LAb. of Protein Engineering, KRIBB

Introduction

Human genome sequencing project has been finished and post-genomic era is already begun. In the wonderland of complete sequences, there is much that genomics cannot do, and so the future belongs to proteomics: the analysis of complete complement of proteins. Now systematic efforts are currently under way to construct  protein microarrays for the analysis of protein expression level.  Protein-protein interactions have been widely used to functional study of unknown genes or proteins. Now it is considered as a new approach to functional genomics, drug discovery, and insight of cancer and diseases. We are interested in the development and application of techniques for protein microarray, especially for in vitro studies of protein function and for application of biochip in diagnostics. Development of protein chip systems provides a sensitive, multipurpose, effective and easy technology to determine protein interactions to protein, nucleic acid, and ligands.  Protein chip could analyze hundreds of proteins in a single experiment,  so it is a very effective method to screen proteins as drug targets in cancer and other human diseases. Since technologies of electronics and protein microarray are used in  the construction of protein chip, cooperation of KRIBB and ETRI in expert system, is on the way for the effective study of it. We immobilized proteins onto chemically derivatized glass slides coated with gold, or silicone wafer at high spatial densities. The proteins attached covalently to the slide surface yet retained their conformation and ability to interact specifically with other proteins in solution. We will develop protein microarrays for  applications such as screening for protein-protein interactions, identifying the substrates of protein kinases and phosphatases, and identifying the protein targets of small molecules.  A high-precision robot designed to manufacture complementary DNA microarrays will be used also to spot proteins onto slides.

Materials and methods

Slide glasses were cleaned and hydrated by acid wash (5% HNO3) at 80℃-90℃ for 60 min followed by rinsing with distilled water. For silanization of slide glass, 1% (v/v) of □-aminopropyltriethoxysilane in acetone was used. Silanized slide glass was dried at room temperature for 5 min and then heated at 115℃ overnight.  The baked slide glass was dipped in 2.5% solution of aldehyde in 0.05M Na2HPO4 buffer adjusted to pH 7.0. The slide glass was dried and used for printing of protein. Dye conjugated antibody or antibody were spotted onto slide glass and kept for overnight in a humid chamber. 1% of BSA solution in PBS was used for masking of unreacted functional groups. The slide glasses having printed antibody was scanned with laser scanner.

Results and discussion

3.1. Immobilization of antibody by alkylamine coupling

Coupling of protein onto slide glass was depend on hydration and cleaning of slide glass so treatment with diluted nitrate was the first step of protein immobilization. Without hydration the amount of immobilized protein was very low. We chose alkylamine coupling method with aldehyde because it is simple, gentle, and rapid. FITC-conjugated antibody was used to test the effectiveness of alkylamine coupling method. FITC-conjugated antibody was spotted onto slide glass and incubated in humid chamber at 37℃ for overnight. After masking with BSA and washing with PBS twice the slide glass was scanned with laser scanner. 1-2 h reaction time was short for the immobilization of antibody but 12 h was quite enough to coupling of antibody.

3.2 Binding activity of immobilized antibody to its antigen

GFP protein was used to test the retaining of protein integrity of immobilized protein onto slide glass because of its fluorescent properties. GFP antibody was immobilized onto slide glass with the same method described previously. After masking of slide glass it was submerged into the PBS solution containing cell lysate having expressed GFP for 1 h at 37℃. The slide glass was rinsed with PBS containing 0.1% Tween 20 three times and scanned. The results showed that GFP antibody retained its conformation on the surface of glass and can interact with GFP protein successfully. Now we are trying to immobilized HBV preS antigen for the detection of preS antibody in the blood. For the microarraying of antibody Q-bot was tried for microspotting of antibody. The quick drying of microspot was main problem of this method and detection of signal was failed. In Figure 1, slides after each step were visualized by laser scanner and compared.

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A                 B                 C                 D

E                 F                G                 H

Fig. 1. A. slide after silanization , B. weakly immobilized FITC-anti-IgG, C. slide1 which failed in immobilization of protein markers, D. slide2 which failed in immobilization of protein markers, E. slide1 which succeeded in immobilization of protein markers, F. slide 2 which succeeded in immobilization of protein markers, G, immobilized Rhodamin- anti -Ig G, H. visualized GFP which was bound to immobilized anti-GFP