Nucleic Acid Programmable Protein Arrays: Versatile Tools for Array‐Based Functional Protein Studies

Abstract

Protein microarrays offer a global perspective on the function of expressed gene products. However, technical issues related to the stability and dynamic range of microarrays printed with purified protein have hampered their widespread adoption. Taking an alternate approach, the Nucleic Acid Programmable Protein Array (NAPPA) is constructed by spotting protein-encoding plasmid DNA at high density, in addressable fashion, on an array surface. Proteins are subsequently generated in situ just prior to experimentation using cell-free expression systems. As such, the NAPPA platform offers a unique and viable alternative that circumvents many of the inherent limitations of spotted protein arrays, enabling diverse functional protein studies including protein-small molecule, protein-protein, antigen-antibody, and protein-nucleic acid interactions. It further offers a versatile and adaptable platform amenable to a variety of capture modalities and expression systems, and, most importantly, construction of the array is accessible to any lab with an array printer and laser slide scanner. This unit is intended to provide a reference for investigators wishing to generate arrays based on this platform, and details (1) the basic construction of cDNA-based protein microarrays from DNA isolation to printing and development, (2) quality-control efforts taken to ensure the usefulness and integrity of microarray data, and (3) a particular example of the application of self-assembling protein arrays to screen for blood-borne antibody biomarkers.

High-throughput identification of proteins with AMPylation using self-assembled human protein (NAPPA) microarrays

“…We have widely published detailed protocols for producing NAPPA arrays, as well as published on recent improvements in the method 21,35–37 . In addition, we have published a video tutorial for how to build NAPPA arrays (https://www.youtube.com/watch?v=Ur1fg9jQv04) 36 . Furthermore, researchers who do not have direct access to resources to build, print, screen, scan, or analyze their own arrays can make use of our central plasmid repository (https://dnasu.org/) and NAPPA core service and facility (http://nappaproteinarray.org/), which distribute plasmid cDNAs and NAPPA arrays at academic prices, respectively 28,38 .…”

Developing aerosol vaccines for Mycobacterium tuberculosis: Workshop proceedings

“…Results of this study demonstrated that antibodies against one arabinomannan fragment were absent in the TB patients but present in the uninfected healthcare worker. In the second antigen microarray study, a nucleic acid programmable protein array (NAPPA) was created in which all 4000 Mtb genes were printed on an array surface and used to generate proteins using a cellfree expression system [30]. The NAPPA protein array was then probed with sera from latently infected controls and TB patients with and without HIV co-infection from the United States and South Africa.…”

Mass Spectrometry and Florescence Analysis of Snap-Nappa Arrays Expressed Using E. coli Cell_Free Expression System

“…Moreover, HCIVT showed a robust lot-to-lot reproducibility. In immune assays, the signals of many antigens were detected only in HCIVT-expressed arrays, mainly due to the reduction in the background signal and the increased levels of protein on the array [14,35]. The protein yields obtained through PURE system has then been matched to that obtained with this innovative cell free IVTT system.…”