Expression of a biotin acceptor peptide-containing protein with potential incorporation on the lentiviral envelope as a viral surface engineering platform

MH Etemadzadeh, A Arashkia F Roohvand, R Ahani, N Mohajel, V Baniasadi, D Norouzian, K Azadmanesh

Abstract


Lentiviral vectors are among the promising viral based-vectors in gene therapy applications, but the efficiency of their targeting needs to be improved. (Strept)avidin-biotin adaptor system is a novel approach to modify the lentiviral envelope for better targeting properties. Herein, we describe utilization of this adaptor system by designing a candidate envelope protein-bearing biotin acceptor peptide (BAP) and evaluation of its expression in 293T cells. To this end, a DNA sequence containing flexible linkers, a 15-aminoacids BAP and specific membrane regions of a viral protein was designed and synthesized in tandem. The synthesized gene was amplified with polymerase chain reaction to include BglII and SalI restriction sites and subcloned into the same sites of pDisplay vector in frame with HA-tag and myc epitope to construct the pDis-GS-BAP. 293T cells were transfected with pDis-GS-BAP and expression of resulting protein (dis-GS-BAP) was evaluated by Western blotting using anti-HA tag antibody. Efficiency of transfection procedure was evaluated by pEGFP-N1 vector and tracking for green fluorescent protein expression via fluorescence microscopy. Restriction analysis and DNA sequencing confirmed the precision of cloning steps. Fluorescence microscopy indicated above 70% transfection efficiency and Western blot analysis of pDis-GS-BAP-transfected 293T cells showed a protein band of approximately 17 kDa corresponding to the predicted size of dis-GS-BAP protein. These promising results indicate the possibility of cell surface expression and further biotinylation of dis-GS-BAP protein in ongoing studies.


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