LaBaer Lab: Technology Development
In this project, we are coupling two technologies (NAPPA and SPRi) that will result in a high-throughput platform to detect and characterize a variety of protein interactions using a label-free detection system that is sensitive, quantitative and provides information on binding kinetics.
A powerful method for studying protein function in high throughput is the application of high-density microscopic arrays displaying numerous proteins. Historically, such arrays have been produced by printing proteins purified from various heterologous cells. Considerable challenges, however, accompany the use of purified protein for printing microarrays. Variable yields of protein result in dynamic ranges in the amount of protein displayed that cover several orders of magnitude, depending on protein size, hydrophobicity and other properties. Batch-to-batch variation may affect experimental reproducibility and the folding and function of some proteins may also be lost during purification, printing and storage. To address this, we invented a novel form of protein microarray, called nucleic acid programmable protein array (NAPPA; U.S. Patent No. 6,800,453). In lieu of producing and printing purified proteins, our method substitutes the printing of cDNAs encoding the proteins. Thus, the resulting array is a DNA array that can be converted into a protein array by adding cell free protein synthesis machinery. This obviates the need to purify proteins, produces human proteins in a mammalian milieu, and avoids concerns about protein stability on the array because the proteins are made just-in-time for assay. Moreover, the method displays a broad variety of proteins, insensitive to protein class or size with a high yield of protein per feature while maintaining a narrow range of protein yield from protein to protein (Science. 2004 305:86; Nat Methods. 2008 5:535).
NAPPA arrays can be used to study protein-protein interactions, protein-drug interactions, search for enzyme substrates, and as tools to search for disease biomarkers. In particular, recent experiments have focused on using these protein microarrays to search for autoantibody responses in cancer patients. Several bona fide autoantibody responses, such as responses to p53, have been detected, and a pilot study of responses to 7500 full length human proteins in 50 breast cancer patients and 50 controls has identified over 700 candidate proteins with more frequent responses in patients. These experiments show promise in finding antibody responses that appear in only cancer patients (J Proteome Res. 2008 7:1490; Proteomics Clin. Appl. 2008.