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Team:Trieste/parts/5
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This construct is designed for the expression of an already descibed engeneered antinorovirus (NoV) monoclonal antibody (mAb 54.6) in fusion with LPP-OmpA. | This construct is designed for the expression of an already descibed engeneered antinorovirus (NoV) monoclonal antibody (mAb 54.6) in fusion with LPP-OmpA. | ||
The antibody is expressed in a small immuno protein (SIP) format containing light (VL) and heavy (VH) variable domains separeted by a flexible peptide linker and constant domain (CH3) of heavy chain of human immunoglobulin A (IgA). It has already been reported that the scFv 54.6 (which compones SIP) binds a native recombinant NoV particles (VLPs) and inhibits VLP interaction with cells. Alpha isotype CH3 domain homodimerizes confering bivalent binding properties to the antibody. | The antibody is expressed in a small immuno protein (SIP) format containing light (VL) and heavy (VH) variable domains separeted by a flexible peptide linker and constant domain (CH3) of heavy chain of human immunoglobulin A (IgA). It has already been reported that the scFv 54.6 (which compones SIP) binds a native recombinant NoV particles (VLPs) and inhibits VLP interaction with cells. Alpha isotype CH3 domain homodimerizes confering bivalent binding properties to the antibody. | ||
| - | LPP-OmpA functions as a leader sequence and an anchor to display the scFv ot the bacterial surface.< | + | LPP-OmpA functions as a leader sequence and an anchor to display the scFv ot the bacterial surface. |
| - | + | <br/> | |
| + | <br/> | ||
The construct consistes of T5 Lac Operator (Bba_K875002), ribosomal binding site, LPP-OmpA, scFv 54.6 antinorovirus, Hystidine tag (6HIS), Terminator (B0015). | The construct consistes of T5 Lac Operator (Bba_K875002), ribosomal binding site, LPP-OmpA, scFv 54.6 antinorovirus, Hystidine tag (6HIS), Terminator (B0015). | ||
| + | <br/> | ||
| + | <br/> | ||
| + | The expression of chimeric protein LPP-OmpA-scFv 54.6-His is regulated by T5 Lac Operator (Bba_K875002). When the promoter T5LacO is induced with IPTG (1mM), the fusion protein LPP-OmpA-scFv 54.6-His is expressed extracellularly on the bacterial surface. LPP-OmpA introduces itself into outer membrane displaying extracellularly antibody attached on itc C-terminus. The number of scFv attached on the bacterial surface, when regulated by this promoter, is estimated up to 30 000*. | ||
| + | This protein has 45.19 kDa. | ||
| + | |||
| + | |||
| + | |||
| + | The construct was tested in E.coli W3110 strain which was previously trasformed with p-REP 4 encoding for the T5 Lac Repressor. The recombinant bacterial colonies were induced at O.D.= 0.4 (2x102 bacterial cells/ml) with IPTG (1mM) at 37°C in shaker. 2ml of bacterial culture was centrifucated and the pellet was resuspended in 200μl of lysis buffer. The samples were then sonificated and boiled for 5 min at 95°C. 10μl of lysates of induced, non-induced and non trasformed bacterial coltures were resolved on SDS-PAGE. The expression of fusion protein LPP-OmpA-scFv 54.6-His was tested by Western blotting with anti-6HIS antibodies (Fig. 2). | ||
| + | |||
| + | |||
| + | Western blot with anti-6HIS antibodies showed the band corresponding to SIP 54.6 at the expected position in the IPTG-induced sample. In the non-induced sample, a weaker signal is also detected suggesting that the promoter is leaky; aspecific signals are also visible. Some of them are due to proteins partially degraded. | ||
| + | |||
| + | Reference: | ||
| + | |||
| + | 1. “Transport and anchoring of 8-lactamase to the external surface of | ||
| + | Escherichia coli” J. A. Francisco, C. F. Earhart and G. Georgiou. | ||
| + | Proc. Natl. Acad. Sci. USA Vol. 89, pp. 2713-2717, April 1992 Biochemistry. | ||
| + | |||
| + | 2. “Recombinant norovirus-specific scFv inhibit virus-like particle binding to | ||
| + | cellular ligands” K. Ettayebi and M. E. Hardy. | ||
| + | Published: 31 January 2008 in Virology Journal 2008, 5:21 | ||
| + | |||
</p> | </p> | ||
| Line 24: | Line 47: | ||
<p class="fancy"> | <p class="fancy"> | ||
<h2>Results</h2> | <h2>Results</h2> | ||
| + | The cloning success has been verified by Colony PCR. (Fig. 1) | ||
| + | The construct has been completely sequenced. | ||
| + | <br/> | ||
<img src="https://static.igem.org/mediawiki/2012/2/27/Trieste_OmpA-SIP.png" alt="Agarose gel"/> | <img src="https://static.igem.org/mediawiki/2012/2/27/Trieste_OmpA-SIP.png" alt="Agarose gel"/> | ||
</p> | </p> | ||
| + | |||
<h2>Modelling</h2> | <h2>Modelling</h2> | ||
</br> | </br> | ||
Revision as of 18:41, 25 September 2012
The Jolly JoCare
a safe probiotic platform for protein expression
BBa_K875005
More
Description
This construct is designed for the expression of an already descibed engeneered antinorovirus (NoV) monoclonal antibody (mAb 54.6) in fusion with LPP-OmpA. The antibody is expressed in a small immuno protein (SIP) format containing light (VL) and heavy (VH) variable domains separeted by a flexible peptide linker and constant domain (CH3) of heavy chain of human immunoglobulin A (IgA). It has already been reported that the scFv 54.6 (which compones SIP) binds a native recombinant NoV particles (VLPs) and inhibits VLP interaction with cells. Alpha isotype CH3 domain homodimerizes confering bivalent binding properties to the antibody. LPP-OmpA functions as a leader sequence and an anchor to display the scFv ot the bacterial surface.The construct consistes of T5 Lac Operator (Bba_K875002), ribosomal binding site, LPP-OmpA, scFv 54.6 antinorovirus, Hystidine tag (6HIS), Terminator (B0015).
The expression of chimeric protein LPP-OmpA-scFv 54.6-His is regulated by T5 Lac Operator (Bba_K875002). When the promoter T5LacO is induced with IPTG (1mM), the fusion protein LPP-OmpA-scFv 54.6-His is expressed extracellularly on the bacterial surface. LPP-OmpA introduces itself into outer membrane displaying extracellularly antibody attached on itc C-terminus. The number of scFv attached on the bacterial surface, when regulated by this promoter, is estimated up to 30 000*. This protein has 45.19 kDa. The construct was tested in E.coli W3110 strain which was previously trasformed with p-REP 4 encoding for the T5 Lac Repressor. The recombinant bacterial colonies were induced at O.D.= 0.4 (2x102 bacterial cells/ml) with IPTG (1mM) at 37°C in shaker. 2ml of bacterial culture was centrifucated and the pellet was resuspended in 200μl of lysis buffer. The samples were then sonificated and boiled for 5 min at 95°C. 10μl of lysates of induced, non-induced and non trasformed bacterial coltures were resolved on SDS-PAGE. The expression of fusion protein LPP-OmpA-scFv 54.6-His was tested by Western blotting with anti-6HIS antibodies (Fig. 2). Western blot with anti-6HIS antibodies showed the band corresponding to SIP 54.6 at the expected position in the IPTG-induced sample. In the non-induced sample, a weaker signal is also detected suggesting that the promoter is leaky; aspecific signals are also visible. Some of them are due to proteins partially degraded. Reference: 1. “Transport and anchoring of 8-lactamase to the external surface of Escherichia coli” J. A. Francisco, C. F. Earhart and G. Georgiou. Proc. Natl. Acad. Sci. USA Vol. 89, pp. 2713-2717, April 1992 Biochemistry. 2. “Recombinant norovirus-specific scFv inhibit virus-like particle binding to cellular ligands” K. Ettayebi and M. E. Hardy. Published: 31 January 2008 in Virology Journal 2008, 5:21
Assembly
Obtained by synthesis
Results
The cloning success has been verified by Colony PCR. (Fig. 1) The construct has been completely sequenced.
Modelling
Looking forward
Link to the Registry
