Protein targeting to lipid bodies
A protein and targeting technology, applied to polypeptides containing positioning/targeting motifs, using vectors to introduce foreign genetic material, bacterial peptides, etc., can solve the three-dimensional structure, factors and motifs of lacquer saponin proteins that have not been reported. Wait for the question
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
preparation example Construction
[0192] b) Preparation of electroporation competent cells
[0193] Plasmids were transferred to Rhodococcus opacus PD630 and M. 2 155 in. For Rhodococcus opacus PD630, prepare electroporation competent cells as described by Kalscheuer et al. [10], for Mycobacterium smegmatis mc 2 155. Preparation of electroporation competent cells was performed as described by Snapper et al. [23].
[0194] c) Preparation of crude cell extract, soluble fraction and TAG content
[0195] Cells of Rhodococcus opacus and M. smegmatis were grown in MSM with reduced ammonium concentrations as described above, collected by centrifugation (20 minutes, 6000×g, 4° C.) and resuspended in two volumes of 0.1 M sodium phosphate buffer (pH 7. 5) in. After passing through the French press (1000MPa) three times, the crude extract was obtained. To obtain the soluble fraction, cellular debris was removed from the crude extract by centrifugation at 16000xg for 30 minutes at 4°C, followed by centrifugation at 1...
Embodiment A1
[0246] Example A1: phaP1 in M. smegmatis mc 2 Distribution of expression and translation products in subcellular fractions in recombinant strains of 155 and Rhodococcus opacus PD630
[0247] To determine the heterologous expression of egfp, phaP1 and phaP1-egfp in recombinant actinomycetes, crude cell extracts from cells grown for 72 h under ammonium-reduced conditions were analyzed by SDS-PAGE and Western blotting as described in the Methods section and cell part. Electropherograms of M. smegmatis cells harboring pJAM2::phaP1 showed an additional protein with an apparent molecular weight of 25 kDa when induced with 0.5% (w / v) acetamide. This molecular weight (MW) corresponds well to the calculated molecular weight of His6-tagged PhaP1. His6-tagged PhaP1 was readily recognized on corresponding Western blots using anti-PhaP1 IgG. However, the synthesis of His6-tagged PhaP1 was significantly less compared to the strain synthesizing the 52kDaPhaP1-eGFP fusion and the unfused 2...
Embodiment A2
[0250] Example A2: PhaP1-eGFP fusion protein in recombinant Rhodococcus opacus PD630 and Mycobacterium smegmatis mc 2 Distribution in 155.
[0251] To confirm the association of PhaP1-eGFP with TAG inclusions in Rhodococcus opacus, by fluorescence microscopy, under conditions grown in Std1 medium and also allowing TAG accumulation when formation of large intracellular TAG inclusions occurs in the cytoplasm, The distribution of the fusion protein was studied in cells grown in ammonium-reduced MSM for 24, 48 and 72 hours. The fluorescence of the fusion proteins is predominantly associated with the TAG content at all stages of their formation. Whereas in cells grown in Std1 medium, the fluorescence was associated with nascent TAG inclusions at the plasma membrane, whereas in cells grown in ammonium-reduced MSM for 24, 48, and 72 hours it was predominantly associated with mature TAG inclusions in the cytoplasm matter binding ( image 3 A-D). Fluorescence was also present to th...
PUM
| Property | Measurement | Unit |
|---|---|---|
| molecular weight | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More