Team:Trieste/parts/4

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<h2>Description </h2>
<h2>Description </h2>
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 single chain fragment variable (scFv) format containing light (VL) and heavy (VH) variable domains separeted by a flexible peptide linker. It has already been reported that the scFv 54.6 binds a native recombinant NoV particles (VLPs) and inhibits VLP interaction with cells. LPP-OmpA functions as a leader sequence and an anchor to display the scFv ot the bacterial surface.
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 single chain fragment variable (scFv) format containing light (VL) and heavy (VH) variable domains separeted by a flexible peptide linker. It has already been reported that the scFv 54.6 binds a native recombinant NoV particles (VLPs) and inhibits VLP interaction with cells. LPP-OmpA functions as a leader sequence and an anchor to display the scFv ot the bacterial surface.
 +
<br/>
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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*.
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This protein  has 45.19 kDa.
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<br/>
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</p>
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</p>
</p>
<h2>Results</h2>
<h2>Results</h2>
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The cloning success has been verified by Colony PCR. (Fig. 1)
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The construct has been completely sequenced.
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 +
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:
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1. “Transport and anchoring of 8-lactamase to the external surface of
 +
    Escherichia coli” J. A. Francisco, C. F. Earhart and G. Georgiou.
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    Proc. Natl. Acad. Sci. USA Vol. 89, pp. 2713-2717, April 1992 Biochemistry.
 +
 +
2. “Recombinant norovirus-specific scFv inhibit virus-like particle binding to     
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    cellular ligands” K. Ettayebi and M. E. Hardy.
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    Published: 31 January 2008 in Virology Journal 2008, 5:21
</br>  
</br>  
<h2>Modelling</h2>
<h2>Modelling</h2>

Revision as of 18:33, 25 September 2012

BBa_K875004

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 single chain fragment variable (scFv) format containing light (VL) and heavy (VH) variable domains separeted by a flexible peptide linker. It has already been reported that the scFv 54.6 binds a native recombinant NoV particles (VLPs) and inhibits VLP interaction with cells. LPP-OmpA functions as a leader sequence and an anchor to display the scFv ot the bacterial surface.
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.

Assembly

Obtained by synthesis.

Results

The cloning success has been verified by Colony PCR. (Fig. 1) The construct has been completely sequenced. 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

Modelling


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