codon-optimized gene for expression in e. coli of full-length algx pp  (Bio Basic Canada)

Journal: The Journal of Biological Chemistry

Article Title: Pseudomonas aeruginosa AlgF is a protein–protein interaction mediator required for acetylation of the alginate exopolysaccharide

doi: 10.1016/j.jbc.2023.105314

Figure Lengend Snippet: ITC analyses of AlgF Pp 29-215 protein–protein interactions with AlgJ Pp 75-370 and AlgX Pp . Protein–protein interactions were investigated by binary titrations of AlgJ:AlgF ( left ), AlgF:AlgX ( center ), and AlgJ:AlgX ( right ) using ITC. Data were fit and analyzed with the Origin software package as shown, and K d values were determined. The AlgJ:AlgX interaction ( right ) was too weak to be analyzed.isothermal titration calorimetry.

Article Snippet: The codon-optimized gene for expression in E. coli of full-length AlgX Pp included flanking Nde I and Xho I restriction sites at the 5′ and 3′ ends, respectively, was synthesized by BioBasic.

Techniques: Protein-Protein interactions, Software, Isothermal Titration Calorimetry

Journal: The Journal of Biological Chemistry

Article Title: Pseudomonas aeruginosa AlgF is a protein–protein interaction mediator required for acetylation of the alginate exopolysaccharide

doi: 10.1016/j.jbc.2023.105314

Figure Lengend Snippet: AlgX-AlgF and AlgI-AlgJ interact in Pseudomonas aeruginosa . A , co-IP from whole-cell lysates of P. aeruginosa expressing VSV-G–tagged AlgX as the bait. Proteins applied to the anti-VSV-G co-IP resin (input, in) and the elution from the resin after washing (immunoprecipitate) were analyzed by Western blot using AlgX- and AlgF-specific antibodies. A strain expressing untagged AlgX was used as a negative binding control. B , analysis of co-IP eluates from the experiment described in panel A by ESI-MS. Spectral counts were the average of six independent co-IP experiments using AlgX C-VSV-G as the bait. Only alginateiosynthetic proteins identified with a minimum of one spectral count in this analysis are listed. C , Western blot analysis of whole-cell lysates of the indicated P. aeruginosa strains using AlgJ-specific antibodies. Antisera recognizing the β-subunit of RNA polymerase was used as a loading control. Co-IP, coimmunoprecipitation; ESI-MS, electrospray ionization mass spectrometry; VSV-G, vesicular stomatitis virus glycoprotein.

Article Snippet: The codon-optimized gene for expression in E. coli of full-length AlgX Pp included flanking Nde I and Xho I restriction sites at the 5′ and 3′ ends, respectively, was synthesized by BioBasic.

Techniques: Co-Immunoprecipitation Assay, Expressing, Western Blot, Binding Assay, Control, Mass Spectrometry, Virus

Journal: The Journal of Biological Chemistry

Article Title: Pseudomonas aeruginosa AlgF is a protein–protein interaction mediator required for acetylation of the alginate exopolysaccharide

doi: 10.1016/j.jbc.2023.105314

Figure Lengend Snippet: Model of the AlgKXF Pp complex involved in alginate acetylation and export. A , AlphaFold2 model of the Pseudomonas putida AlgKXF complex (AlgK Pp , green ; AlgX Pp , periwinkle; AlgF Pp , light blue ). B , surface representation of the AlgKXF Pp complex with the AlgX Pp active site highlighted in yellow . C , AlgF Pp residues involved in the interaction interface with AlgX Pp . The model of AlgF Pp is colored by secondary structure (coil, light blue ; strand; blue ; helix, dark blue ). Residues involved in hydrogen bonding interactions, salt bridge interactions, or hydrogen bonding and salt bridge interactions are represented by blue, red, and yellow text , respectively. Residues that are underlined represent side chain interactions and residues that are italicized represent main chain interactions. In both panels the N terminiand C termini are represented by N and C, respectively.

Article Snippet: The codon-optimized gene for expression in E. coli of full-length AlgX Pp included flanking Nde I and Xho I restriction sites at the 5′ and 3′ ends, respectively, was synthesized by BioBasic.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Pseudomonas aeruginosa AlgF is a protein–protein interaction mediator required for acetylation of the alginate exopolysaccharide

doi: 10.1016/j.jbc.2023.105314

Figure Lengend Snippet: Model of alginate O -acetylation machinery. AlgI receives an acetyl group from an as-yet unknown donor in the cytoplasm and the acetate group is passed through the inner membrane and transferred to AlgJ. AlgF mediates interactions in the periplasm allowing for the acetate to be passed from AlgJ to AlgX, where AlgX O -acetylates the newly polymerized mannuronic acid. OM, PG, IM denote the outer membrane, peptidoglycan, and inner membrane, respectively. Orange arrow indicates how the acetyl group is transferred between AlgI, AlgJ, and AlgX.

Article Snippet: The codon-optimized gene for expression in E. coli of full-length AlgX Pp included flanking Nde I and Xho I restriction sites at the 5′ and 3′ ends, respectively, was synthesized by BioBasic.

Techniques: Membrane