Development of repeatable arrays of proteins using immobilized DNA microplate (RAPID-M) technology.

Nur Suhanawati Ashaari,Rahmah Mohamed,Suganti Ramarad,Sheila Nathan,Yuka Hara,Dzulaikha Khairuddin,Nor Muhammad Mahadi,Nor Azurah Mat Akhir

doi:10.1186/s13104-015-1637-3

Abstract

BackgroundProtein microarrays have enormous potential as in vitro diagnostic tools stemming from the ability to miniaturize whilst generating maximum evaluation of diagnostically relevant information from minute amounts of sample. In this report, we present a method known as repeatable arrays of proteins using immobilized DNA microplates (RAPID-M) for high-throughput in situ protein microarray fabrication. The RAPID-M technology comprises of cell-free expression using immobilized DNA templates and in situ protein purification onto standard microarray slides.ResultsTo demonstrate proof-of-concept, the repeatable protein arrays developed using our RAPID-M technology utilized green fluorescent protein (GFP) and a bacterial outer membrane protein (OmpA) as the proteins of interest for microarray fabrication. Cell-free expression of OmpA and GFP proteins using beads-immobilized DNA yielded protein bands with the expected molecular sizes of 27 and 30 kDa, respectively. We demonstrate that the beads-immobilized DNA remained stable for at least four cycles of cell-free expression. The OmpA and GFP proteins were still functional after in situ purification on the Ni–NTA microarray slide.ConclusionThe RAPID-M platform for protein microarray fabrication of two different representative proteins was successfully developed.

Highlights

Protein microarrays have enormous potential as in vitro diagnostic tools stemming from the ability to miniaturize whilst generating maximum evaluation of diagnostically relevant information from minute amounts of sample
During development of the RAPID-M platform, the modified T7 terminator (T7Term) primer was mixed with the immobilization beads (Fig. 1a) to create the beads-immobilized T7Term primer (Fig. 1b) in the polymerase chain reaction (PCR) microwell plate
Separate protein purification is not necessary in the RAPID-M approach as on-chip protein purification was incorporated onto the microarray slides

Summary

Introduction

Protein microarrays have enormous potential as in vitro diagnostic tools stemming from the ability to miniaturize whilst generating maximum evaluation of diagnostically relevant information from minute amounts of sample. The RAPID-M technology comprises of cell-free expression using immobilized DNA templates and in situ protein purification onto standard microarray slides. The production of protein arrays has relied on cellular expression, purification and immobilization of individual proteins onto solid supports. This pipeline is a laborious and time-consuming process that inevitably presents a fresh set of technical challenges. To address these problems, several platforms have been developed that use cell-free systems and immobilized DNA to create protein arrays such as Nucleic Acid Programmable Protein Array (NAPPA) [7], Protein in situ Array (PISA) [8], DNA Array to Protein Array (DAPA)

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Conclusion