A cross-cultural study of domestic luminous environment in the United Kingdom and Japan

Abstract not available

Flow cytometry assay for InaK-Ns and InaK-N/ARG1s.

<p>Cells containing different vectors labeled with Ddylight649-conjugated antibody against the HA epitope tag were analyzed by flow cytometry. The excitation laser was 638nm, and the emission filter was 660/20 BP. A-M indicated cells containing the pET23a-T empty vector; pET23a-InaK-N vector; pET23a <i>-</i>ssMalE-InaK-N vector; pET23a-ssTorA-InaK-N vector; pET23a-D₆-InaK-N vector; pET23a-E₆-InaK-N vector; pET23a-K₆-InaK-N vector; pET23a-InaK-N/ARG1 vector; pET23a-ssMalE-InaK-N/ARG vector; pET23a-ssTorA-InaK-N/ARG1 vector; pET23a-D₆-InaK-N/ARG1 vector; pET23a-E₆-InaK-N/ARG1 vector; and pET23a-K₆-InaK-N/ARG1 vector, respectively.</p

Surface Immobilization of Human Arginase-1 with an Engineered Ice Nucleation Protein Display System in <i>E</i>. <i>coli</i>

<div><p>Ice nucleation protein (INP) is frequently used as a surface anchor for protein display in gram-negative bacteria. Here, MalE and TorA signal peptides, and three charged polypeptides, 6×Lys, 6×Glu and 6×Asp, were anchored to the N-terminus of truncated INP (InaK-N) to improve its surface display efficiency for human Arginase1 (ARG1). Our results indicated that the TorA signal peptide increased the surface translocation of non-protein fused InaK-N and human ARG1 fused InaK-N (InaK-N/ARG1) by 80.7% and 122.4%, respectively. Comparably, the MalE signal peptide decreased the display efficiencies of both the non-protein fused InaK-N and InaK-N/ARG1. Our results also suggested that the 6×Lys polypeptide significantly increased the surface display efficiency of K₆-InaK-N/ARG1 by almost 2-fold, while also practically abolishing the surface translocation of non-protein fused InaK-N, indicating the interesting roles of charged polypeptides in bacteria surface display systems. Cell surface-immobilized K₆-InaK-N/ARG1 presented an arginase activity of 10.7 U/OD₆₀₀ under the optimized conditions of 40°C, pH 10.0 and 1 mM Mn²⁺, which could convert more than 95% of L-Arginine (L-Arg) to L-Ornithine (L-Orn) in 16 hours. The engineered InaK-Ns expanded the INP surface display system, which aided in the surface immobilization of human ARG1 in <i>E</i>. <i>coli</i> cells.</p></div

The chemical reagents used in this study.

<p>The chemical reagents used in this study.</p

Fluorescence microscope assay of InaK-N/ARG1s.

<p>Surface fluorescence of the cells harboring various InaK-N/ARG1s under a fluorescence microscope with the excitation laser of 488nm, and the EGFP detection channel being used. A-G indicated cells containing empty pET23a-T vector; pET23a-InaK-N/ARG1 vector; pET23a-ssMalE-InaK-N/ARG1 vector; pET23a-ssTorA-InaK-N/ARG1 vector; pET23a-D₆-InaK-N/ARG1 vector; pET23a-E₆-InaK-N/ARG1 vector; and pET23a-K₆-InaK-N/ARG1 vector, respectively. 1: detecting of FITC signal, 2: the bright field, 3: the merge of FITC signal and bright field.</p

Constructs in the study.

<p>(A) Different N-terminal fusions of InaK-N. (B) Different InaK-Ns fused with C-terminal human ARG1. (C) Schematic diagram of the cloning method for making the constructs. (D) <i>InaK-Ns</i> PCR products. Lanes 1–6 are PCR products for <i>InaK-N</i>, <i>ssMalE</i>-<i>InaK-N</i>, <i>ssTorA</i>-<i>InaK-N</i>, <i>D</i>_<i>6</i>-<i>InaK-N</i>, <i>E</i>_<i>6</i>-<i>InaK-N</i>, and <i>K</i>_<i>6</i>-<i>InaK-N</i>. (E) PCR products for <i>InaK-N/ARG1s</i>. Lane 1–8 <i>InaK-N</i>, <i>human ARG1</i>, <i>InaK-N/ARG1</i>, <i>ssMalE</i>-<i>InaK/ARG1</i>, <i>ssTorA</i>-<i>InaK-N/ARG1</i>, <i>D</i>_<i>6</i>-<i>InaK-N/ARG1</i>, <i>E</i>_<i>6</i>-<i>InaK-N/ARG1</i>, and <i>K</i>_<i>6</i>-<i>InaK-N/ARG1</i>. M: DNA Ladder.</p

Immobilization of the ARG1 fusions to the cell outer membrane.

<p>(A) SDS-PAGE and Western-Blot analysis of the human ARG1 fusions on the outer membrane. Upper panel is the SDS-PAGE analysis and the lower panel is the western-blot analysis. M: protein ladder; T: total cell proteins; OM: outer membrane proteins. InaK-N/ARG1; ssTorA-InaK-N/ARG1; K₆-InaK-N/ARG1; E₆-InaK-N/ARG1; D₆-InaK-N/ARG1; and ssMalE-InaK-N/ARG1 presented the recombinant strain expressing these fusion proteins, respectively; Control presented the recombinant strain containing empty pET23a-T. (B) Enzyme activity and proteinase K accessibility of the InaK-N/ARG1s. Proteinase K-: recombinant strain was untreated with proteinase K; Proteinase K+: recombinant strain was treated with proteinase K. K6-InaK-N/ARG1; E6-InaK-N/ARG1; D6-InaK-N/ARG1; ssMalE-InaK-N/ARG1; K6-InaK-N/ARG1 presented the recombinant strains expressing these fusion proteins, respectively.</p

Characterization of the cell surface-immobilized InaK-N-E/ARG1s.

<p>(A) Relative activity of the cells bearing K₆-InaK-N/ARG1 at different pH values. (B) Relative activity of the cells bearing K₆-InaK-N/ARG1 at different temperatures. (C) Operational stability of the cells bearing K₆-InaK-N/ARG1. The maximum activity was normalized to 100% in all of the assays. (D) MC trace analysis of the hydrolysis of molecular standard L-Orn. (E) MC trace analysis of the hydrolysis of molecular standard L-Arg.(F) MC trace analysis of the hydrolysis of L-Arg catalyzed by the cells bearing K₆-InaK-N/ARG1.</p

Sequences of the export signals and polypeptides used in this study.

<p>Sequences of the export signals and polypeptides used in this study.</p

Whole cell enzyme activity.

<p>Whole cell enzyme activity.</p