Bacterial production and binding assessment of a novel streptavidin-TAT fusion-based delivery vehicle
Bacterial production and binding assessment of a novel streptavidin-TAT fusion-based delivery vehicle
Aref Farokhi-Fard,1Fatemeh Davami,2,*
1. Biotechnology research center, Pasteur institute of Iran, Tehran, Iran 2. Biotechnology research center, Pasteur institute of Iran, Tehran, Iran
Introduction: The cytoplasmic membrane is a barrier to uptake of many therapeutic macromolecules. Different strategies proposed to overcome this barrier are mostly afflicted by low efficiencies and/or cytotoxicity. A promising strategy for intracellular delivery of large cargos is the use of cell-penetrating peptides (CPPs). TAT peptide (GRKKRRQRRR) derived from Human immunodeficiency virus type 1 (HIV-1) is one of the most efficient CPPs and also a nuclear localization signals (NLSs). The fusion of TAT with tetrameric streptavidin (TAT-tSA, a 60 kDa fusion protein) combines the biotin binding ability of tSA and internalization activity of TAT to form a versatile platform for intracellular and/or nuclear delivery of various biotinylated cargos. Despite the rapid and efficient internalization into living cells, most of the TAT-tSA molecules have been shown to be entrapped inside the endocytic vesicles in previous studies. Here, we designed a novel delivery platform: fusion of TAT and monomeric streptavidin (mSA) equipped with a human derived fusogenic peptide, S19. High endosomolytic activity of S19 could be served to solve the endosomal entrapment problem of previously described TAT-tSA. This report is about the bacterial expression, purification and preliminary functional characterization of mSA-S19-TAT fusion.
Methods: The sequence of mSA was extracted from cas9-mSA/sgRNA vector by polymerase chain reaction (PCR). TAT and S19 were prepared as synthetic single stranded oligonucleotide. The oligos were phosphorylated and hybridized to form phosphorylated dsDNA fragments with sticky ends. All the fragments were ligated, PCR amplified and then cloned in pET-32a (C2) vector at NcoI and XhoI restriction sites. Hence, mSA-S19-TAT coding fragment was cloned in frame with upstream thioredoxin (Trx)-tag and carboxy terminus His-tag of the vector. Confirmed plasmid was transformed to chemically competented expression hosts, Origami B strain. A single colony of Origami B/C2 was cultured in LB broth medium supplemented with 4.7 mM biotin and induced in a soluble favorable condition: 0.05 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), 20 °C and 200 rpm for overnight. SDS PAGE was used to analyze the expression of fusion and purified protein (315 AA, 34.167 KDa). NiNTA affinity chromatography (Qiagen resin) was accomplished for purification of recombinant protein in native condition using. The purified protein was concentrated and then was buffer exchanged to 50 mM Tris-HCL, pH 8 by use of centrifugal amicon filter (10 KDa cut off). Protein concentration was estimated with spectrophotometer (using NanoDrop: Abs280nm, at extinction coefficient of 50,420 M-1 cm-1). The biotin binding ability of fusion was evaluated in triplicate by enzyme-linked immunosorbent assay (ELISA) using a plate pre-coated with biotinylated bovine serum albumin (B-BSA) and HRP conjugated polyclonal anti-His-tag antibody.
Results: PCR, restriction digestion and sequencing were used for confirmation of cloning procedure. The fusion was successfully expressed as a soluble Trx-fusion entity without any observable toxicity on host. The solubly expressed fusion was successfully purified using NiNTA affinity chromatography and eluted fusion was concentrated and extensively buffer exchanged to 0.1 M Glycine-HCL, pH 2.3 to remove any bonded biotins. The purified concentrated proteins then were buffer exchanged to 50 mM Tris-HCL, pH 8. The final production yield after buffer exchanging and concentration was more than 6 mg soluble fusion per 1 liter culture. The fusion exhibited excellent biotin binding ability in ELISA assay in seven deferent concentrations: OD450nm value of 3.010 and 0.225 at concentrations of 150 µg/ml (the highest concentration) and 2.3 µg/ml (the lowest concentration) of the produced fusion respectively.
Conclusion: The mSA we used, has much smaller size in comparison to tSA and reversible biotin binding property. Due to the presence of a structurally important intramolecular disulfide bond as well as aggregation propensity of streptavidin, we used Origami B/pET32a system to avoid cumbersome in vitro refolding. We extensively buffer exchanged the purified fusion in acidic condition to separate the bonded biotin and obtain active protein. Based on ELISA results, S19 and TAT did not interfere with biotin binding activity of mSA moiety. More characterizations is being done to investigate the cell penetration and endosomal escape ability of the proposed delivery system.