BBa_K875004

More

Description

This construct is designed for the expression of an already described engineered 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 construct consist 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 the outer membrane displaying extracellularly the antibody attached on its C-terminus.
This protein has 45.19 kDa.

Assembly

The sequence LPP-OmpA-scFv is obtained by PelB-ScFv construct after replacement of the leader sequences. Then through cloning we created the final construct.

Results

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

Gel LPP-OmpA-scFv

FIG.1 Electrophoresis in gel 1% agarose whit ethidium bromide of T5LacO-LPP-OmpA-scFv-6His-TT. Fragment LPP-OmpA-scFv-6His-TT, ,previously double digested with XbaI/PstI, was then cloned downstream the T5LacOperator in plasmid pSB1C3 double digested with SpeI/PstI.

The construct was tested in E.coli W3110 strain which was previously trasformed with p-REP 4 encoding for the Lac Repressor. The recombinant bacterial culture was induced at O.D.= 0.4 (2x108 bacterial cells/ml) with IPTG (1mM) at 37°C in shaker. 2ml of bacterial culture were centrifuged and the pellet was resuspended in 200μl of lysis buffer. The samples were then sonicated and boiled for 5 min. 10μl of lysates of induced, non-induced and non trasformed bacterial cultures 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 LPP-OmpA-scFv

FIG. 2. Expression of scFv 54.6 cloned in fusion with the LPP-OmpA leader sequence . Western blots of lysates of E.coli W3110 bacterial strain expressing the recombinant protein scFv 54.6 (45,19KDa), induced or non-induced with IPTG. The blot was reacted with the monoclonal F24-796 anti-6XHIS antibody.

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 partially degraded protein.

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