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Revision as of 23:56, 13 October 2014

Results / Heterologous Expression

To examine whether the T4MBP was heterologous expressed by plants, leaves of Nicotiana tabacum plants were injected with pORE-E3_2x25S_T4MBP containing Rhizobium radiobacter. After separation of the leaf extracts by SDS-PAGE, an immunostaining aimed to specifically detect the T4MBP´s internal FLAG-tag (Figure 1A). Attributed to the antibodie´s unspecifity, the T4MBP could not be detected in the plant leaf extracts. Hence, the T4MBP coding construct was transferred into the pASK plasmid adding an n-terminal Strep-tag and a c-terminal 6xHistidine-tag to the target protein. Based on the engineered pASK_T4MBP, the heterologous expression and detection of the T4MBP could be successfully achieved in the end.

Labwork 1

• Cloning the synthesized GeneArt construct into our pORE-E3_2x35S vector system
• Infiltration of Nicotiana tabacum leaves with pORE-E3_2x25S_T4MPB-containing Rhizobium radiobacter cells.
1. Extraction of proteins from the leaf tissue and subsequent seperation via SDS-PAGE (VERWEIS).
2. Transfer onto a PVDF membrane and immunostaining(VERWEIS)via anti-FLAG-tag antibody.

Results

Immunostaining of Nicotiana tabacum leave extracts after infiltrating them with pORE-E3_2x35S_T4MBP-containing Rhizobium radiobacter solution (T4MBP). Due to a failed immunologic detection, no difference between the samples infiltrated by water (control) and by bacteria solution were observed (1A). Another repetition A final volume of 15 µl of leave extracts was separated via 12 % SDS-PAGE, proteins were transferred onto a PVDF membrane and decorated by an anti-FLAG-tag antibody. Black arrows indicate the expected molecular weight of the T4MBP.The standard (M) used here is the Spectra Multicolor Broad Range Protein Ladder.

As the host for heterologous T4MBP expression we chose the Origami2 cells to work with. This E. coli strain expresses huge amounts of cytosolic disulfide isomerase cytosolic and thus elevates the chance of disulfide formation for recombinant proteins. To improve the quality of proteins, furthermore, we lowered the expression temperatures to 16 °C.

Labwork 2

• Cloning the metal-binding-sequences into the pASK plasmid
• Heterologous Expression and Detection of T4MBP.
1. Lysis of bacteria cells and protein precipitation by TCA.
2. Analysis by SDS-PAGE (VERWEIS).
3. Transfer of separated proteins onto a PVDF membrane and an immunostaining (VERWEIS) by an anti-6xHistidine-tag antibody.

Results

Figure 2: Immunostaining of cytosolic extracts from pASK_T4MBP containing Origami2TM cells (T4MBP). While the control (c) shows no reaction, the T4MBP medium elucidated a specific signal at a molecular weight of ~40 kDa. A final volume of 15 µl of prepared proteins was separated by 12 % SDS-PAGE. Proteins were than transferred onto a PVDF membrane and decorated by an anti-6xHistidine-tag antibody. A black arrow indicates the expected molecular weight. The standard (M) used here was the Spectra Multicolor Broad Range Protein Ladder.