Phillip C Wright

Publications (154) View all

• Article: Inverse metabolic engineering to improve Escherichia coli as an N-glycosylation host.

  Jagroop Pandhal, Lauren B A Woodruff, Stephen Jaffe, Pratik Desai, Saw Y Ow, Josselin Noirel, Ryan T Gill, Phillip C Wright
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  ABSTRACT: An inverse metabolic engineering strategy was used to select for Escherichia coli cells with an increased capability to N-glycosylate a specific target protein. We developed a screen for E. coli cells containing extra-chromosomal DNA fragments for improved ability to add precise sugar groups onto the AcrA protein using the glycosylation system from Campylobacter jejuni. Four different sized (1, 2, 4 and 8 kb) genomic DNA libraries were screened, and the sequences that conferred a yield advantage were determined. These advantageous genomic fragments were mapped onto the E. coli W3110 chromosome. Five candidate genes (identified across two or more libraries) were subsequently selected for forward engineering verification in E. coli CLM24 cells, utilizing a combination of internal standards for absolute quantitation and pseudo-selective reaction monitoring (pSRM) and Western blotting validation. An increase in glycosylated protein was quantified in cells overexpressing 4-α-glucantransferase and a phosphoenolpyruvate-dependent sugar phosphotransferase system, amounting to a 3.8-fold (engineered cells total = 5.3 mg L(-1) ) and 6.7-fold (engineered cells total = 9.4 mg L(-1) ) improvement compared to control cells, respectively. Furthermore, increased glycosylation efficiency was observed in cells overexpressing enzymes involved with glycosylation precursor synthesis, enzymes 1-deoxyxylulose-5-phosphate synthase (1.3-fold) and UDP-N-acetylglucosamine pyrophosphorylase (1.6-fold). To evaluate the wider implications of the engineering, we tested a modified Fc fragment of an IgG antibody as the target glycoprotein with two of our engineered cells, and achieved a ca. 75% improved glycosylation efficiency. Biotechnol. Bioeng. © 2013 Wiley Periodicals, Inc.
  Biotechnology and Bioengineering 04/2013; · 3.95 Impact Factor
• Article: The quantitative proteomic response of Synechocystis sp. PCC6803 to phosphate acclimation.

  Matthew A Fuszard, Saw Yen Ow, Chee Sian Gan, Josseilin Noirel, Nigel G Ternan, Geoff McMullan, Catherine A Biggs, Kenneth F Reardon, Phillip C Wright
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  ABSTRACT: BACKGROUND: Inorganic phosphate (Pi) is a critical nutrient for all life and is periodically limiting in marine and freshwater provinces, yet little is understood how organisms acclimate to fluctuations in Pi within their environment. To investigate whole cell adaptation, we grew Synechocystis sp. PCC6803, a model freshwater cyanobacterium, in 3%, and 0.3% inorganic phosphate (Pi) media. The cells were allowed to acclimate over 60 days, and cells were harvested for quantitative high throughput mass spectrometry-based proteomics using the iTRAQTM labelling technology. RESULTS: In total, 120 proteins were identified, and 52 proteins were considered differentially abundant compared to the control. Alkaline phosphatase (APase) activities correlated significantly (p < 0.05) with observed relative PhoA abundances. PstS1 and PstS2 were both observed, yet PstS1 was not differentially more abundant than the control. Phycobilisome protein abundances appeared to be coordinated, and are significantly less abundant in 0.3% Pi than 3% Pi cultures. Also, the central metabolic cell function appears to have shifted towards the production of (NADPH) reducing energy and nucleotide sugars. CONCLUSIONS: This acclimation response bears strong similarity to the previously reported response to nitrogen deprivation within Synechocystis sp. PCC 6803. However, it also demonstrates some characteristics of desiccation stress, such as the regulation of fatty acids and increased abundance of rehydrin in the 3% Pi culture.
  Aquatic biosystems. 02/2013; 9(1):5.
• Article: Quantitative proteomic analysis of the exoelectrogenic bacterium Arcobacter butzleri ED-1 reveals increased abundance of a flagellin protein under anaerobic growth on an insoluble electrode.

  Ana G Pereira-Medrano, Matthew Knighton, Gregory J S Fowler, Zi Yen Ler, Trong Khoa Pham, Saw Yen Ow, Andrew Free, Bruce Ward, Phillip C Wright
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  ABSTRACT: Exoelectrogens have the ability to generate electricity in mediator-less microbial fuel cells (MFCs) by extracellular electron transfer to the anode. We investigate the anode-specific responses of Arcobacter butzleri ED-1, the first identified exoelectrogenic Epsilonproteobacterium. iTRAQ and 2D-LC MS/MS driven proteomics were used to compare protein abundances in A. butzleri ED-1 when generating an electronegative potential (-225mV) in an anaerobic half-cell - either growing as an electrogenic biofilm or suspended in the liquid medium - versus an microaerobic culture. This is the first quantitative proteomic study concentrating on growth of an exoelectrogen during current generation. From 720 proteins identified and quantified (soluble and insoluble sub-proteomes), statistical analysis reveals 75 differentially-expressed proteins. This dataset was enriched in proteins regulating energy and intermediary metabolism, electron and protein transport. Flagellin up-regulation was concomitant with electron transport in the anodic cells, while decreased abundance of a methyl-accepting chemotaxis protein suggested that flagella were involved in communication with the anode surface and electrogenesis, rather than motility. Two novel cytochromes potentially related to electron transport were up-regulated in anaerobic cultures. We demonstrate that employing an insoluble extracellular electron acceptor for anaerobic growth regulates multiple proteins involved in cell surface properties, electron transport and the methylcitrate cycle.
  Journal of proteomics 10/2012; · 5.07 Impact Factor
• Article: Bioinformatic study of the relationship between protein regulation and sequence properties.

  Xin Zou, Trong Khoa Pham, Phillip C Wright, Josselin Noirel
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  ABSTRACT: Although protein expression and regulation have been intensively studied, a complete picture of its mechanisms is still to be drawn. Analysis of high-throughput quantitative proteomics data provides a way to better understand protein regulation. Here, we introduce a bioinformatic analysis method to correlate protein regulation with individual amino acid patterns. We compare the amino acid composition between groups of regulated and unregulated proteins and investigate the correlation between codon usage patterns and protein regulation levels in two Sulfolobus species in "biofilm vs planktonic" experiments. The identified amino acids can then be associated with the regulation of specific gene functions. Strikingly, our analysis shows that functional categories of regulated proteins with similar composition and codon usage pattern of specific amino acids behave similarly. This finding can contribute to a better understanding of protein and gene expression regulation and could find applications in gene optimisation.
  Genomics 07/2012; 100(4):240-4. · 3.02 Impact Factor
• Article: An integrated workflow for extraction and solubilization of intermediate filaments from colorectal biopsies for proteomic analysis.

  Debabrata Majumdar, Ria Rosser, Suzanne Havard, Alan J Lobo, Phillip C Wright, Caroline A Evans, Bernard M Corfe
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  ABSTRACT: We report a technique for isolation and solubilization of intermediate filament (IF) proteins from colonic biopsies compatible with both gel electrophoresis and liquid chromatography "shotgun" proteomics using mass spectrometry (MS). This is important because changes in the IF proteome, particularly in keratin expression and modification, are noted in colonic mucosa of patients with colorectal cancer. Though keratins have traditionally been dissolved in high concentration of urea, the latter solvent precludes efficient proteolytic digestion by trypsin prior to gel-free LC-MS/MS approaches. The extraction of cytoskeletal proteins was initially evaluated using MCF-7 cancer cell lines using a published, differential detergent solubilization protocol. IF proteins were extracted from colonic biopsies using a combination of homogenization and sonication. Since comparable efficiency of solubilization was noted on the extracted IF from cell lines between urea and guanidine hydrochloride (GuHCl) in triethylammonium bicarbonate buffer, isolated proteins from endoscopic biopsies were solubilized in GuHCl. Using immunoblotting techniques, we successfully demonstrated isolation of keratins and preservation of posttranslational modifications (phosphorylation, acetylation). Dissolved proteins were tryptically digested and peptides analyzed by MS, showing the functionality of the workflow in shotgun proteomic applications, specifically compatibility of the workflow for isobaric tagging relative and absolute quantification based quantitation approaches.
  Electrophoresis 07/2012; 33(13):1967-74. · 3.30 Impact Factor