Robust microarray production of freshly expressed proteins in a human milieu

Festa, Fernanda; Rollins, Sean M.; Vattem, Krishna M.; Hathaway, Margarita; Lorenz, Phillip; Mendoza, Eliseo A.; Yu, Xiaobo; Qiu, Ji; Kilmer, Greg; Jensen, Penny; Webb, Brian; Ryan, Ed T.; LaBaer, Joshua

doi:10.1002/prca.201200063

Cited by 30 publications

(46 citation statements)

References 32 publications

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“…In cell-free protein arrays, a nucleotide template is printed on the slide and used to produce proteins in vitro with cell-free expression systems from several organisms such as E. coli, wheat germ, and rabbit reticulocyte lysate, etc. (24,25). These proteins can be engineered to contain fusion tags that enable their capture to the array surface with an appropriate agent.…”

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confidence: 99%

Copper-catalyzed azide-alkyne cycloaddition (click chemistry)-based Detection of Global Pathogen-host AMPylation on Self-assembled Human Protein Microarrays

Woolery

Luong

et al. 2014

Molecular & Cellular Proteomics

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AMPylation (adenylylation) is a recently discovered mechanism employed by infectious bacteria to regulate host cell signaling. However, despite significant effort, only a few host targets have been identified, limiting our understanding of how these pathogens exploit this mechanism to control host cells. Accordingly, we developed a novel nonradioactive AMPylation screening platform using high-density cell-free protein microarrays displaying human proteins produced by human translational machinery. We screened 10,000 unique human proteins with Vibrio parahaemolyticus VopS and Histophilus somni IbpAFic2, and identified many new AMPylation substrates. Two of these, Rac2, and Rac3, were confirmed in vivo as bona fide substrates during infection with Vibrio parahaemolyticus. We also mapped the site of AMPylation of a non-GTPase substrate, LyGDI, to threonine 51, in a region regulated by Src kinase, and demonstrated that AMPylation prevented its phosphorylation by Src. Our results greatly expanded the repertoire of potential host substrates for bacterial AMPylators, determined their recognition motif, and revealed the first pathogen-host interaction AMPylation network. This approach can be extended to identify novel substrates of AMPylators with different domains or in different species and readily adapted for other post-translational modifications. Molecular & Cellular

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confidence: 99%

Copper-catalyzed azide-alkyne cycloaddition (click chemistry)-based Detection of Global Pathogen-host AMPylation on Self-assembled Human Protein Microarrays

Woolery

Luong

et al. 2014

Molecular & Cellular Proteomics

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“…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.…”

Section: Fluorescence Analysismentioning

confidence: 99%

“…It is known [14] that HCIVT performs better than RRL IVTT. The yield of protein synthesized in HCIVT is more than 10 times higher than RRL.…”

Section: Fluorescence Analysismentioning

confidence: 99%

“…The printed slides were expressed using a reconstituted E. coli coupled cell-free expression system (E. coli IVTT) (PURExpress in vitro system, New England Biolabs, Ipswich, MA, USA) [14,31]); briefly, slides were blocked in SuperBlock (Thermo Scientific, Rockford, IL, USA) for 1hour at room temperature with constant agitation and dried with filtered air. HybriWells gaskets (Grace Biolabs, Bend, OR, USA) were applied on the top of the slides and 160μl of E. coli IVTT, prepared according to the manufacturers' instructions, was added.…”

Section: Production and Expression Of Nappamentioning

confidence: 99%

“…The addition of a SNAP tag to each protein enabled its capture to the array through an anti-SNAP antibody printed simultaneously with the expression plasmid [14].…”

Section: Introductionmentioning

confidence: 99%

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

Nicolini

Spera

Festa³

et al. 2013

J Nanomed Nanotechnol

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We present the analysis of an innovative kind of self-assembling protein microarray, the "Nucleic Acid Programmable Protein Array" (NAPPA), express with the SNAP tag in E.coli coupled self free expression system. The goal is to develop a standardize procedure to analyze the protein protein interaction occurred on NAPPA array combining label free Mass Spectrometry (MS) and fluorescence technology for protein microarray. We employ in the process "Protein synthesis Using Recombinant Elements" (PURE) system. For the first time an improved version of NAPPA, that allows for functional proteins to be synthesized in situ -with a SNAP tag -directly from printed cDNAs just in time for assay, has been expressed with a novel cell-free transcription/translation system reconstituted from the purified components necessary for Escherichia coli translation -the PURE system -and analyzed both in fluorescence and in a label free manner by four different mass spectrometers, namely three Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF), a Voyager, a Bru ker Autoflex and a Bruker Ultraflex, and Liquid Chromatography-Electrospray Ionization Mass Spectrometry (LC-ESI-MS/MS). Due to the high complexity of the system, an ad hoc bioinformatic tool has been needed to develop for their successful analysis. The contemporary fluorescence analysis of NAPPA, expressed by means of PURE system, has been performed to confirm the improved characterization of this new NAPPA-SNAP system.

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Overview of Cell‐Free Protein Synthesis: Historic Landmarks, Commercial Systems, and Expanding Applications

Chong

2014

CP Molecular Biology

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During early days of molecular biology, cell-free protein synthesis played an essential role in deciphering the genetic code and contributed to our understanding of translation of protein from messenger RNA. Owning to several decades of major and incremental improvements, modern cell-free systems have achieved higher protein synthesis yields at lower production costs. Commercial cell-free systems are now available from a variety of material sources, ranging from “traditional” E. coli, rabbit reticulocyte lysate and wheat germ extracts to recent insect and human cell extracts to defined systems reconstituted from purified recombinant components. Though each cell-free system has certain advantages and disadvantages, the diversity of the cell-free systems allows in vitro synthesis of a wide range of proteins for a variety of downstream applications. In the post-genomic era, cell-free protein synthesis has rapidly become the preferred approach for high throughput functional and structural studies of proteins and a versatile tool for in vitro protein evolution and synthetic biology. This article provides a brief history of cell-free protein synthesis and describes key advances in modern cell-free systems, practical differences between widely used commercial cell-free systems, and applications of this important technology.

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Robust microarray production of freshly expressed proteins in a human milieu

Cited by 30 publications

References 32 publications

Copper-catalyzed azide-alkyne cycloaddition (click chemistry)-based Detection of Global Pathogen-host AMPylation on Self-assembled Human Protein Microarrays

Copper-catalyzed azide-alkyne cycloaddition (click chemistry)-based Detection of Global Pathogen-host AMPylation on Self-assembled Human Protein Microarrays

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

Overview of Cell‐Free Protein Synthesis: Historic Landmarks, Commercial Systems, and Expanding Applications

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