Atery wall binding peptide-linked high mobility grop box-1 peptide for DNA delivery to smooth muscle cells

Abstract

HMGB1(high mobility group box1), a nuclear protein, are able to bind and condense with plasmid DNA. HMGB1 are composed of 3 domains. They contain 2 basic HMG boxes A and B, and a highly acidic C-terminal domain. Previously we generated a truncated HMGB1 derivative which lacks the acidic C-tail, reported to decrease the affinity of HMG proteins for DNA, and HMG box B. HIV-TAT protein was conjugated to truncated formation of HMGB1(HMGB1A) to enhance transfection efficiency. The artery wall is an important target organ for treatment of cardiovascular disease by non-viral gene therapy. Apolipoprotein B-100 contains many receptor binding domain, including artery wall binding domain. In this study, artery wall binding peptide (ABP) was linked to TAT-HMGB1A for artery wall targeting and it enhance gene expression. Recombinant his tag fusion protein, TAT-HMG1A-ABP were cloned and expressed in Escherichia coli at high yield. This TAT-HMGB1A-ABP protein was purified by overexpression of plasmid constructs using Nickel affinity resins. The complexes formation between TAT-HMGB1AABP and plasmid DNA was confirmed by gel retardation assay at various weight ratios. The band of pCMV-Luc was retarded as the amount of TAT-HMGB1A-ABP increased. Above 2/1 weight ratio protein carrier and DNA was markedly retarded in the 1% agarose gel, indicating effective DNA binding and complex formation. pCMV-Luc and peptide complexes were added to A7R5 smooth muscle cells in serum free medium for 4 h. After 4 h media was changed. Luciferase activities were determined 24 hr later. At an weight ratio of 5, 10, 15, 20, 25 and 30 the transfection efficiency of theTAT-HMGB1AABP/pDNA was evaluated, and highest transfection efficiency was at a 20/1 weight ratio. PLL was used as a control carrier and compared to transfection efficiency of TAT-HMGB1A-ABP. PLL/DNA complexes was prepared at a 2/1 weight ratio. TAT-HMGB1AABP/pDNA complex had higher efficiency than PLL. MTT assay was performed to investigate whether protein carrier is non-toxic. Until presence of 20 ug of TAT-HMGB1A-ABP in 24 well plate, the protein did not have toxicity. The Recombinant protein for artery wall targeting has considerable potential to facilitate DNA delivery to smooth muscle cells.