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Latest revision as of 21:53, 17 October 2014

Results / Heavy metals / 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, immunostaining experiments aimed to specifically detect the T4MBP´s internal Flag-tag (Figure 1). 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 tag change, the pASK derived T4MBP could be successfully expressed in the end (Figure 2).

Plant based Expression

• 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 separation via SDS-PAGE.
2. Transfer onto a PVDF membrane and immunostaining via anti-Flag-tag antibody.

Results

Figure 1: 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 (1B) 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 bacterial host for the heterologous T4MBP production we chose Origami 2 cells to work with. This E. coli strain expresses huge amounts of cytosolic disulfide isomerase and thus raises the disulfide bond formation for recombinant proteins. Furthermore, to improve the quality of proteins, we lowered the expression temperature to 16 °C.

E.coli based Expression

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

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