title: In situ protein microarrays capable of real-time kinetics analysis based on surface plasmon resonance imaging.
abstract: In recent years, in situ protein synthesis microarray technologies have enabled protein microarrays to be created on demand just before they are needed. In this paper, we utilized the TUS-TER immobilization technology to allow label-free detection with real-time kinetics of protein-protein interactions using surface plasmon resonance imaging (SPRi). We constructed an expression-ready plasmid DNA with a C-terminal TUS fusion tag to directionally immobilize the in situ synthesized recombinant proteins onto the surface of the biosensor. The expression plasmid was immobilized on the polyethylene imine-modified gold surface, which was then coupled with a cell-free expression system on the flow cell of the SPRi instrument. The expressed TUS fusion proteins bind on the surface via the immobilized TER DNA sequence with high affinity (∼3-7×10(-13)M). The expression and immobilization of the recombinant in situ expressed proteins were confirmed by probing with specific antibodies. The present study shows a new low cost method for in situ protein expression microarrays that has the potential to study the kinetics of protein-protein interactions. These protein microarrays can be created on demand without the problems of stability associated with protein arrays used in the drug discovery and biomarker discovery fields.
authors: National Center for Nanoscience and Technology, Beijing 100190, People's Republic of China; University of Chinese Academy of Sciences, Beijing 100049, People's Republic of China.
authors: Amita
authors: A
authors: Nand
authors: National Center for Nanoscience and Technology, Beijing 100190, People's Republic of China; University of Chinese Academy of Sciences, Beijing 100049, People's Republic of China.
authors: Vikramjeet
authors: V
authors: Singh
authors: National Center for Nanoscience and Technology, Beijing 100190, People's Republic of China; University of Chinese Academy of Sciences, Beijing 100049, People's Republic of China.
authors: Javier Batista
authors: JB
authors: Pérez
authors: National Center for Nanoscience and Technology, Beijing 100190, People's Republic of China; University of Chinese Academy of Sciences, Beijing 100049, People's Republic of China.
authors: Deependra
authors: D
authors: Tyagi
authors: National Center for Nanoscience and Technology, Beijing 100190, People's Republic of China; University of Chinese Academy of Sciences, Beijing 100049, People's Republic of China.
authors: Zhiqiang
authors: Z
authors: Cheng
authors: National Center for Nanoscience and Technology, Beijing 100190, People's Republic of China; University of Chinese Academy of Sciences, Beijing 100049, People's Republic of China. Electronic address: jizhu@nanoctr.cn.
authors: Jingsong
authors: J
authors: Zhu
mesh_terms: D015374:Biosensing Techniques; D007700:Kinetics; D040081:Protein Array Analysis; D011506:Proteins / Q000032:analysis*; D020349:Surface Plasmon Resonance / Q000379:methods*
chemical_list: D011506:Proteins
keywords: In situ protein microarray; Plasmid DNA; Surface plasmon resonance imaging