Description

This construct is designed for the expression of secreted, already described engineered antinorovirus (NoV) monoclonal antibody (mAb 54.6).
The antibody is expressed in a single chain fragment variable (scFv) format containing light (VL) and heavy (VH) variable domains separated 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. PelB is a leader sequence often used to produce secreted proteins.

The construct consistes of T5 Lac Operator (Bba_K875002), ribosomal binding site, PelB-scFv 54.6 antinorovirus, HIstidine tag (6HIS), Terminator (B0015).

The expression of the protein PelB-scFv 54.6-HIS is regulated by T5 Lac Operator (Bba_K875002). When the promote T5 Lac O is induced with IPTG (1mM), scFv 54.6 is expected to be secreted. PelB drives the protein into the periplasmic space where is clived off. The resulting protein then can be released into extracellular space by passing through porins.

Molecular Weight: 29,25 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 transformed with p-REP 4 coding 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 colture was centrifuged 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 cultures were resolved on SDS-PAGE. The expression of fusion protein 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 scFv 54.6 at the expected position in the IPTG-iduced sample. In the non-induced sample, a weaker signal is also detected suggesting that the promoter is leaky. Aspecific signals are visible also. Some of them are due to proteins partially degraded.

Reference:
1. “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�

Looking forward

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