Journal: PLoS ONE

Article Title: New Infestin-4 Mutants with Increased Selectivity against Factor XIIa

doi: 10.1371/journal.pone.0144940

Figure Lengend Snippet: (A) A web-logo representation of the P 2 –P 5’ binding loop sequences of 89 proteinase inhibitors from the Kazal family . The amino acid positions are numbered by the wt-Inf4 sequence Phe9–Val15. Sequences of the corresponding reactive site fragments from wt-Inf4 and its mutants are shown below. (B) The MD simulation indicated a π-cation interaction (yellow dashed line) in wt-Inf4 between the phenyl group of Phe9 and the guanidinium group of Arg10. The nitrogen atoms are shown in blue, oxygen in red, and hydrogen in white. The torsion angles ψ, ϕ, χ 1 , and χ 2 that describe the scissile bond conformation and rotations of the Arg10 side chain are drawn. (C ) Superposition (main chain atoms) of wt-Inf4 (the most abundant conformation over the MD trajectory) with the structures of Inf4 mutants whose reactive loop solely adopted the canonical conformation. A close-up view of the reactive site region is indicated with a rectangle (wt-Inf4 –cyan, Mutant A–yellow, Mutant B–orange, Mutant C–green, Mutant 15 –magenta; this coloring is also used in the following Figs). The scissile bond of wt-Inf4 is indicated with a wavy line; a star indicates the Arg10 carbonyl oxygen. (D) A Coomassie-stained 12.5% SDS-PAGE showing the one-step purification procedure for one of the Inf4 versions expressed in E . coli BL21(DE3) cells (Trx-Mutant B is shown as an example). The protein samples applied to the gel are as follows: M –molecular weight marker (kDa); Induction 0 h and 4 h –cell lysates before 1 mM IPTG induction and 4 h later, respectively; Debr and Sol –insoluble and soluble fractions, respectively; Fl-thr –flow-through of the Sol fraction applied to the column; Wash –washing the column with 50 mM Tris, pH 7.5, 500 mM NaCl, 100 mM imidazole; Resin –Chelating Sepharose with bound and washed Trx-Mutant B (26 kDa); Elt –Chelating Sepharose after Trx-Mutant B elution with 50 mM Tris-HCl, pH 7.5, 500 mM imidazole; thereafter, the protein sample was applied on the SOURCE 30Q column. (E) Following the two-step purification, the Trx-fused protein was cleaved with bovine thrombin. Thrombin (mU) –indicated amounts of bovine thrombin (0, 1, 2, 3, 4, 6, and 10 milliunits) were added to 10 μg of Trx-Mutant B resulting in two separate bands of Mutant B (13.1 kDa) and Trx (12.9 kDa). (F) Mutant B –indicated quantities of the Mutant B protein ( 10 , 1 , and 0.5 μg loaded into gel) that was purified after the cleavage reaction, as described in the sub-section “Expression of the thioredoxin-fused infestin-4 and its mutants”.

Article Snippet: The target product was collected in the flow-through fraction which was then applied to a Tricorn 10/100 column containing SOURCE 15Q resin (GE Healthcare).

Techniques: Binding Assay, Sequencing, Mutagenesis, Staining, SDS Page, Purification, Molecular Weight, Marker, Expressing

Journal: Journal of Peptide Science

Article Title: An explorative study towards the chemical synthesis of the immunoglobulin G1 Fc CH3 domain

doi: 10.1002/psc.3126

Figure Lengend Snippet: Attempts of stepwise synthesis of [Met(O)‐432]‐IgG1‐Fc 398–450 ( 1 ). A, Assembly attempt on polystyrene‐divinylbenzene 2‐chlorotrityl resin preloaded with glycine (0.63 mmol/g). The RP‐HPLC profiles of the crude peptide acids with free N ‐terminus at cycle 22 (till Cys‐429) and 32 (till Ser‐419, 1a TRT ) were obtained by using method A. B, Assembly attempt on the polar and low‐loaded resin NovaSyn TGT (0.2 mmol/g Fmoc‐glycine) by using three pseudoproline dipeptides (in red). The RP‐HPLC profile of the Fmoc‐protected crude peptide acid at cycle 44 (till Ser‐407, 1a TGT+ψ ) was obtained by using method C with the Nucleosil C‐18 column (MALDI‐TOF‐MS peaks for M + H + and [M + 2H + ]/2. M calc. for C 242 H 352 N 62 O 69 S 2 : 5298.01 Da)

Article Snippet: Analytical RP‐HPLC was performed using a Thermo Fisher Scientific Dionex UltiMate 3000 UHPLC system (Germering, Germany) and either a Syncronics C‐18 column (100 Å, 5 μm, 250 × 4.6 mm, Thermo Fisher Scientific) at a flow rate of 1.5 mL/min or a Nucleosil C‐18 column (100 Å, 5 μm, 250 × 4 mm, Macherey‐Nagel, Düren, Germany) at a flow rate of 1 mL/min.

Techniques: