api restriction  (Reddit Inc)

Journal: PLoS ONE

Article Title: Phage Display of the Serpin Alpha-1 Proteinase Inhibitor Randomized at Consecutive Residues in the Reactive Centre Loop and Biopanned with or without Thrombin

doi: 10.1371/journal.pone.0084491

Figure Lengend Snippet: Panel A shows the cloning strategy employed to create T7Select10-3b phage constructs displaying API M358R or libraries displaying API with randomized RCL codons. 1) The API M358R cDNA was mobilized from pBAD-H 6 API M358R by PCR that appropriately positioned EcoRI (green) and HindIII cohesive ends, and ensured that it was in-frame for subsequent insertion into the T7 Select 10-3b vector, in plasmid pUC19-API M358R. 2) Degeneracy was introduced at either P2–P1 or P7–P3 codons using a sense primer overlapping the unique PmlI site or a degenerate antisense (NNN primer) overlapping the unique SauI site. PCR products were inserted into phosphatase-treated PmlI- and SauI-restricted pUC19-API M358R and the resulting plasmid library biologically amplified in E. coli TOP10. 3) EcoRI-and HindIII-restricted total digestion products of the P2–P1 or P7–P3 plasmid libraries, respectively, were then ligated to T7Select10-3b vector arms. 4) The recombinant T7Select10-3b library, containing either the API P2–P1 randomized or the API M358R P7–P3 randomized inserts, was then packaged into phages to create the respective phage display libraries. Pink, light blue, yellow, and purple API-encoding inserts (between steps 2 and 3) indicate the encoding of different variants, and the correspondingly coloured phages (step 4) represent their displayed products. Panel B shows the biopanning procedure. 1) A phage lysate produced by infection of E. coli BLT5615 with a T7Select10-3b API library was reacted with thrombin in solution (green squares), which bound to some displayed sequences (e.g. blue but not yellow or pink). 2) A biotinylated antibody specific to thrombin (Y-shaped symbols) was added to the thrombin-phages mixture, reacting with thrombin bound to phages via API-thrombin serpin-enzyme complexes. 3) Streptavidin-coated magnetic beads (yellow S shown on red circular symbols) were added to the mixture, reacting preferentially with antibody-thrombin-phage complexes. 4) Magnetic bead-streptavidin-thrombin-phage complexes were concentrated using a magnet. 5) After washing, bead assemblies were used to directly infect a fresh culture of E. coli BLT5615 to start the next round of biopanning.

Article Snippet: PmlI-SauI restricted pUC19-API M358R was treated with calf intestinal alkaline phosphatase (New England Biolabs, Pickering, ON) to preclude self-ligation of any singly cut molecules.

Techniques: Cloning, Construct, Plasmid Preparation, Amplification, Recombinant, Produced, Infection, Magnetic Beads

Journal: PLoS ONE

Article Title: Phage Display of the Serpin Alpha-1 Proteinase Inhibitor Randomized at Consecutive Residues in the Reactive Centre Loop and Biopanned with or without Thrombin

doi: 10.1371/journal.pone.0084491

Figure Lengend Snippet: Panel A: 1×10 10 pfu of purified T7Select10-3b API M358R (API M358R phages) and T7Select10-3b S-tag phages (Control phage) were separately reacted with (+) or without (−) 20 nM thrombin. Reactions were solubilized with SDS, electrophoresed on SDS-polyacrylamide gels under reducing conditions, immunoblotted, and probed with an affinity-purified sheep anti-thrombin antibody. Molecular weight marker positions, in kDa, are indicated to the left of the figure. The left panel represents four contiguous lanes of a single immunoblot. The right panel shows the reaction of 20 nM thrombin with 8.5 nM purified, soluble API M358R or saline controls; both lanes were derived from the same immunoblot but were not contiguous on the original image. Panel B: T7Select10-3b API M358R phages and T7Select10-3b S-tag phages were combined 1:100 and reacted with 0.5 nM thrombin for times shown on the x axis, prior to biopanning with biotinylated anti-thrombin IgG and streptavidin-linked magnetic beads. Washed beads were used to infect E. coli and aliquots of the resulting lysates used to form plaques on agarose/agar plates. Nitrocellulose plaque lifts were probed with anti-API antibodies to determine the percentage of immunoreactive plaques as a fraction of the total, shown on the y axis. “No IIa” (open bar) refers to a sample from the original mixture of phages subjected to plaque assay directly, without biopanning.

Article Snippet: PmlI-SauI restricted pUC19-API M358R was treated with calf intestinal alkaline phosphatase (New England Biolabs, Pickering, ON) to preclude self-ligation of any singly cut molecules.

Techniques: Purification, Control, Affinity Purification, Molecular Weight, Marker, Western Blot, Saline, Derivative Assay, Magnetic Beads, Plaque Assay

Journal: PLoS ONE

Article Title: Phage Display of the Serpin Alpha-1 Proteinase Inhibitor Randomized at Consecutive Residues in the Reactive Centre Loop and Biopanned with or without Thrombin

doi: 10.1371/journal.pone.0084491

Figure Lengend Snippet: After biopanning the P2–P1 phage display library for five rounds with (+) or without (−) thrombin, the API inserts from 20 plaque-purified phage were sequenced from each biopanned library. Panel A shows the observed frequency, as a percentage, for variants with the dipeptide sequences shown on the x axis. Black bars correspond to variants identified by thrombin panning, while white bars correspond to those identified by mock selection; hatched bars identify the PP sequence identified in both groups. Panel B shows API variants with the same P2–P1 dipeptide sequences as in Panel A, but transferred to plasmids (pBAD) for expression as soluble, intracellular proteins. Cultures containing plasmids expressing the API P2-P1 dipeptide identified on the x axis were lysed and the extent to which anti-API immunoreactive proteins bound to thrombin immobilized on microtiter plate wells was quantified as the optical density at 450 nm. The mean of duplicate determinations ± SD is shown. As in Panel A, black bars relate to thrombin-panned and white bars to mock-selected candidates. Cross-hatched bar shows the results from a colony transformed with pBAD-H 6 API M358R as a positive control.

Article Snippet: PmlI-SauI restricted pUC19-API M358R was treated with calf intestinal alkaline phosphatase (New England Biolabs, Pickering, ON) to preclude self-ligation of any singly cut molecules.

Techniques: Purification, Selection, Sequencing, Expressing, Transformation Assay, Positive Control

Journal: PLoS ONE

Article Title: Phage Display of the Serpin Alpha-1 Proteinase Inhibitor Randomized at Consecutive Residues in the Reactive Centre Loop and Biopanned with or without Thrombin

doi: 10.1371/journal.pone.0084491

Figure Lengend Snippet: Panel A: Thrombin-selected API RCL inserts from round 5 of phage display were transferred en masse to a plasmid expression library and bacteria were transformed. Eighty colonies were screened by the thrombin capture assay (as shown in ) and the resulting optical density values were normalized to API M358R lysate controls. The mean of two determinations is shown. Panel B: Same as panel A, except that the source of the plasmid expression library was round 5 of mock selection. The horizontal line in both panels highlights the relative optical density ratio of API M358R in the normalized results (y = 1.0).

Article Snippet: PmlI-SauI restricted pUC19-API M358R was treated with calf intestinal alkaline phosphatase (New England Biolabs, Pickering, ON) to preclude self-ligation of any singly cut molecules.

Techniques: Plasmid Preparation, Expressing, Bacteria, Transformation Assay, Selection

Journal: PLoS ONE

Article Title: Phage Display of the Serpin Alpha-1 Proteinase Inhibitor Randomized at Consecutive Residues in the Reactive Centre Loop and Biopanned with or without Thrombin

doi: 10.1371/journal.pone.0084491

Figure Lengend Snippet: P7–P3 variants binding thrombin in a capture assay more effectively than API M358R.

Article Snippet: PmlI-SauI restricted pUC19-API M358R was treated with calf intestinal alkaline phosphatase (New England Biolabs, Pickering, ON) to preclude self-ligation of any singly cut molecules.

Techniques: Binding Assay

Journal: PLoS ONE

Article Title: Phage Display of the Serpin Alpha-1 Proteinase Inhibitor Randomized at Consecutive Residues in the Reactive Centre Loop and Biopanned with or without Thrombin

doi: 10.1371/journal.pone.0084491

Figure Lengend Snippet: Kinetic properties of API M358R variants with P7–P3 substitutions.

Article Snippet: PmlI-SauI restricted pUC19-API M358R was treated with calf intestinal alkaline phosphatase (New England Biolabs, Pickering, ON) to preclude self-ligation of any singly cut molecules.

Techniques:

Journal: PLoS ONE

Article Title: Phage Display of the Serpin Alpha-1 Proteinase Inhibitor Randomized at Consecutive Residues in the Reactive Centre Loop and Biopanned with or without Thrombin

doi: 10.1371/journal.pone.0084491

Figure Lengend Snippet: The second order rate constant (k 2 ; mean of 5 determinations ± SD) was plotted as a function of the optical density at 450 nm in the thrombin capture assay (mean of 2–3 determinations) for API M358R and the 12 P7–P3 variants with kinetic results shown in . The mean OD 450 (n = 5) for mock thrombin capture assays lacking API-related proteins in the lysate (0.05) was taken as indicating a K 2 of 0. The correlation of determination (r 2 ) is shown next to the regression line.

Article Snippet: PmlI-SauI restricted pUC19-API M358R was treated with calf intestinal alkaline phosphatase (New England Biolabs, Pickering, ON) to preclude self-ligation of any singly cut molecules.

Techniques: