The major technological challenges, inspired by nanotechnology, on the development of protein microarrays and chips, protein patterning and biosensor were intensively investigated. This approach makes use of various biological research areas such as molecular self-assembly, spatial positioning, microconstruction, biocomposite fabrication and nanomachines. Therefore, nanobiotechnology, a new discipline that employs biomolecules as building blocks and biomolecular self-assembly as one of the construction methods, offers exciting possibilities for the miniaturization of analytical instruments.
At first, a novel method for manufacturing peptide microarrays by elevating the peptide on the layer of protein by fusion protein approach was carried out. It provided invaluable information elevating peptides on the protein layer of protein allows sensitive, specific, and efficient detection of peptide-protein interactions without the need for complicated chemical modification of solid supports and peptides. It was found that kinase activity could be detected with as low as 0.09 fmol of kemptide, which is about 1000 fold times more sensitive than that 0.1 pmol obtained with other microarray systems. Therefore, this new strategy will not only be useful in high-throughput and cost-effective screening of kinase substrate peptides, but also be generally applicable in studying various protein-peptide interactions.
Secondly, biosensor for kinase activity based on an individual MWCNT nanoelectrode was suggested. Moreover, changes of conductance of substrate peptide coated metallic MWCNTs upon reaction of PKA and dephosphorylation indicate that kinase activity sensor can be constructed at the nano-scale level of an individual MWCNT. In this result, these phenomena have to be continued to demonstrate that individual MWCNTs are ideal nanomaterials for building functional nano devices and biosensors.
Thirdly, recombinant fusion protein that are genetically linked to poly-3-hydroxybutyrate (PHB) depolymerase binding domain as a capture ligand was applied to selective immobilization onto PHB microspheres for immunological assay. These findings encouraged us to apply our knowledge about bead-based assays to a biologically relevant system for the possible diagnosis of severe acute respiratory syndrome (SARS). The immobilization on PHB microspheres is specific, enabling immobilization of the fusion protein without timeconsuming any further purification procedures. Furthermore, the binding between the fusion proteins and PHB microspheres is strong, irreversible and stable under most stringent conditions.
In addition, new example of a combination of μCP and selective immobilization of SBDs-fusion protein for the fabricating biologically active substrates was suggested. Our method is based on the site-selective noncovalent adsorption of SBDs to polyester thin films without loss of biological activity by using μCP method.
Finally, the introduction of gold binding protein (GBP) by genetically engineered techniques is demonstrated to be a viable method to immobilize proteins onto gold surfaces in a controlled manner. With addition of a GBP to the N-terminal end of enhanced green fluorescence protein (EGFP), the fusion protein was densely packed mono-layer onto gold surface. Protein surface coverage was visualized for studying protein morphology at the molecular level by atomic force microscopy (AFM). Microcontact printing (μCP) was used to generate the micropattern of fusion protein and to demonstrate the selective immobilization of proteins and surface plasmon resonance (SPR) imaging was used to visualize the pattern of the immobilized fusion protein. Specific antigen-antibody interaction (EGFP-anti-EGFP) was carried out using surface plasmon resonance (SPR) technique for directly characterizing the bioactivity of the fusion proteins attached to the gold substrates.
유전공학적 기법을 이용하여 기질 펩타이드를 링커 단백질을 매개로 기판에 고정함으로써 단백질 칩을 이용한 반응 단백질과 그의 기질 펩타이드간의 반응분석 방법에 있어 반응 단백질과 그의 기질 펩타이드간의 반응은 칩에 고정된 기질 펩타이드에 효과적으로 반응하여 우수한 검출감도를 나타내었다. 단백질 칩 상에서의 분자량이 작은 펩타이드와 분자량이 큰 효소단백질간 및 펩타이드와 반응항체간의 반응성을 높여 주어, 펩타이드와 단백질간 효과적인 반응분석을 신속하고 대량으로 수행할 수 있도록 함으로써, 펩타이드-단백질 반응에 기초한 효율적이고 경제적인 대량 검색, 생화학적 분석, 신약 후보물질 분석 및 질병 진단 등의 연구와 응용에 크게 기여할 수 있을 것이라 사료된다.
목적단백질 유전자 또는 목적 펩타이드를 코딩하는 핵산에 PHB 에 특이적으로 결합하는 분해효소의 기질결합영역을 발현시키는 유전자를 대장균에 도입, 융합 발현시킨 후, 상기 융합 단백질을 PHB 마이크로스피어에 특이적 (specific)으로 고정되도록 고안하여,목적단백질 유전자를 코딩하는 유전자 말단에 알카리제네스 페칼리스 (Alcaligenes faecalis)의 세포외 PHB 분해효소 (depolymerase) 기질결합영역이 융합 발현되도록 플라스미드를 제작하고, 전기 플라스미드로 대장균을 형질전환하여 배양함으로써 녹색형광단백질에 PHA 분해효소의 기질결합영역이 결합된 융합 단백질을 생성하고, 이를 PHA 마이크로스피어에 특이적 (specific)으로 고정하고, 제작된 마이크로스피어를 이용하여 flow cytometry 분석법을 이용하여 단백질- 단백질 반응을 검출할 수 있는 것을 확인하였고 또한 사스 (Severe Acute Respiratory Syndrome, SARS) 항체 검출법에 이를 응용하였다. 또한 코마모나스 테스토스테로니, 슈도모나스 스튜체리 (Pseudomonas stutzeri, Comamonas testosteroni)의 세포외 PHB 분해효소 (depolymerase) 분해효소의 기질결합영역이 결합된 융합 단백질을 제조하여 이를 응용하여 단백질 패터닝 (patterning), surface plasmon resonance (SPR)을 응용한 분석법 또한 개발하였다.
목적단백질 유전자 말단에 골드 칩에 특이적으로 결합하는 GBP 를 발현시키는 유전자를 융합시키도록 대장균에 도입, 발현시킨 후, 상기 융합 단백질을 골드 칩에 위치 특이적 (site-specific)으로 고정되도록 고안하여 목적단백질 N 말단에 GBP 가 융합 발현되도록 플라스미드를 제작하고, 전기 플라스미드로 대장균을 형질전환하여 배양함으로써 녹색형광단백질에 GBP 가 결합된 융합 단백질을 생성하고, 이를 골드칩에 위치 특이적 (site-specific)으로 고정하여, 단백질 패터닝, SPR, SPR imaging 을 이용하여 단백질-단백질 반응을 검출에 활용하여 바이오 센서에 응용 가능성을 보여 주었다. 이러한 나노생물공학기법을 이용한 생물학적 분석법 개발은 나노생물공학의 핵심적인 플랫포옴 테크놀로지 (platform technology) 를 제공하는데 그 의의가 있다고 할 수 있다.
