Protein–protein interactions: Analysis of a false positive GST pulldown result

Wissmueller, Sandra; Font, Josep; Liew, C.W.; Cram, E.D.; Schroeder, Thilo; Turner, Jeremy; Crossley, Merlin; Mackay, Joel P.; Matthews, Jacqueline M.

doi:10.1002/prot.23068

Cited by 17 publications

(17 citation statements)

References 26 publications

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“…The results from both GST pull-down (Fig 6 F) and co-IP experiments (Fig 6 G) revealed that PRR14-2 peptide, which contains the proline rich domain, binds specifically to GRB2. The binding of C-terminal of PRR14 (PRR14-4) to GRB2 was also detected in GST pull-down but not in Co-IP experiment, suggesting a non-specific binding in GST pull-down analysis 37 . Immunofluorescence staining showed the possibility of the 2 proteins interacting with each other spatially and temporally (S2 B).…”

Section: Resultsmentioning

confidence: 96%

PRR14 is a novel activator of the PI3K pathway promoting lung carcinogenesis

et al. 2016

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Chromosomal focal amplifications often cause an increase in gene copy number contributing to the pathogenesis of cancer. PRR14 overexpression is associated with recurrent locus amplification in lung cancer, and it correlates with a poor prognosis. We show that increased PRR14 expression promoted and reduced PRR14 expression impeded lung cancer cell proliferation. Interestingly, PRR14 cells were markedly enlarged in size and exhibited an elevated activity of PI3-kinase/Akt/m-TOR pathway, which was associated with a heightened sensitivity to the inhibitors of PI3K and mTOR. Biochemical analysis revealed that PRR14, as a proline-rich protein, binds to the Src homology 3 (SH3) domains of GRB2 resulting in PI3K activation. Significantly, 2 cancer patient-derived PRR14 mutants displayed considerably augmented GRB2-binding and enhanced ability of promoting cell proliferation. Together with the in vivo data demonstrating a strong tumor-promoting activity of PRR14 and the mutants, our work uncovered this proline-rich protein as a novel activator of the PI3K pathway that promoted tumorigenesis in lung cancer.

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Section: Resultsmentioning

confidence: 96%

PRR14 is a novel activator of the PI3K pathway promoting lung carcinogenesis

et al. 2016

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“…However, it is often overlooked that the presence of soluble protein from bacterial expression does not guarantee correct folding. Soluble, partially-folded aggregates often form and can bind non-specifically to other proteins [22]. Affinity tags can also impact folding, leading to non-specific interactions [23].…”

Section: Resultsmentioning

confidence: 99%

Refinement of the subunit interaction network within the nucleosome remodelling and deacetylase (NuRD) complex

et al. 2017

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The nucleosome remodelling and deacetylase (NuRD) complex is essential for the development of complex animals. NuRD has roles in regulating gene expression and repairing damaged DNA. The complex comprises at least six proteins with two or more paralogues of each protein routinely identified when the complex is purified from cell extracts. To understand the structure and function of NuRD, a map of direct subunit interactions is needed. Dozens of published studies have attempted to define direct inter-subunit connectivities. We propose that conclusions reported in many such studies are in fact ambiguous for one of several reasons. First, the expression of many NuRD subunits in bacteria is unlikely to lead to folded, active protein. Second, interaction studies carried out in cells that contain endogenous NuRD complex can lead to false positives through bridging of target proteins by endogenous components. Combining existing information on NuRD structure with a protocol designed to minimize false positives, we report a conservative and robust interaction map for the NuRD complex. We also suggest a 3D model of the complex that brings together the existing data on the complex. The issues and strategies discussed herein are also applicable to the analysis of a wide range of multi-subunit complexes.

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“…The Halo-Tag ® and its ligand bind covalently, leading to a strong, irreversible bond [16]. Further, it increases the amount of soluble protein expressed in contrast to other tags [17], as it reduces the formation of inclusion bodies during recombinant protein expression.…”

Section: Introductionmentioning

confidence: 99%

Microarray-based method for screening of immunogenic proteins from bacteria

Hoppe

Bier

Nickisch‐Rosenegk

2012

Journal of Nanobiotechnology

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BackgroundDetection of immunogenic proteins remains an important task for life sciences as it nourishes the understanding of pathogenicity, illuminates new potential vaccine candidates and broadens the spectrum of biomarkers applicable in diagnostic tools. Traditionally, immunoscreenings of expression libraries via polyclonal sera on nitrocellulose membranes or screenings of whole proteome lysates in 2-D gel electrophoresis are performed. However, these methods feature some rather inconvenient disadvantages. Screening of expression libraries to expose novel antigens from bacteria often lead to an abundance of false positive signals owing to the high cross reactivity of polyclonal antibodies towards the proteins of the expression host. A method is presented that overcomes many disadvantages of the old procedures.ResultsFour proteins that have previously been described as immunogenic have successfully been assessed immunogenic abilities with our method. One protein with no known immunogenic behaviour before suggested potential immunogenicity.We incorporated a fusion tag prior to our genes of interest and attached the expressed fusion proteins covalently on microarrays. This enhances the specific binding of the proteins compared to nitrocellulose. Thus, it helps to reduce the number of false positives significantly. It enables us to screen for immunogenic proteins in a shorter time, with more samples and statistical reliability. We validated our method by employing several known genes from Campylobacter jejuni NCTC 11168.ConclusionsThe method presented offers a new approach for screening of bacterial expression libraries to illuminate novel proteins with immunogenic features. It could provide a powerful and attractive alternative to existing methods and help to detect and identify vaccine candidates, biomarkers and potential virulence-associated factors with immunogenic behaviour furthering the knowledge of virulence and pathogenicity of studied bacteria.

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Protein–protein interactions: Analysis of a false positive GST pulldown result

Cited by 17 publications

References 26 publications

PRR14 is a novel activator of the PI3K pathway promoting lung carcinogenesis

PRR14 is a novel activator of the PI3K pathway promoting lung carcinogenesis

Refinement of the subunit interaction network within the nucleosome remodelling and deacetylase (NuRD) complex

Microarray-based method for screening of immunogenic proteins from bacteria

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