Biosynthetic approach for functional protein microarrays

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• 作者：Brian Stamos¹ ; Leticia Loredo¹ ; Subhash Chand ; Tuan V. Phan ; Yanbo Zhang ; Sridev Mohapatra ; Krishnan Rajeshwar ; Roshan Perera ; ^{perera@uta.edu}
• 关键词：Protein microarray ; Recombinant technology ; Proteomics ; Cytochrome P450 ; Enzyme immobilization
• 刊名：Analytical Biochemistry
• 出版年：2012
• 期刊代码：93_00032697
• 类别：imm
• 出版时间：15 May, 2012
• 卷：424
• 期：2
• 页码：114-123
• 文件大小：1879 K

摘要

Protein microarrays have emerged as an indispensable research tool for providing information about protein functions and interactions through high-throughput screening. Traditional methods for immobilizing biomolecules onto solid surfaces have been based on covalent and noncovalent binding, entrapment in semipermeable membranes, microencapsulation, sol gel, and hydrogel methods. Each of these techniques has its own strengths but fails to combine the most important tenets of a functional protein microarray such as covalent attachment, native protein conformation, homogeneity of the protein monolayer, control over active site orientation, and retention of protein activity. Here we present a selective and site-directed covalent immobilization technique for proteins via a benzoxazine ring formation through a Diels-Alder reaction in water and a genetically encoded 3-amino-l-tyrosine (3-NH₂Tyr) amino acid. Fully functional protein microarrays, with monolayer arrangements and complete control over their orientations, were generated using this strategy.