Construction, expression and characterization of a chimeric multi-domain protein mediating specific DNA transfer.
The delivery of plasmid DNA to target cells using a simple, defined, non-viral system is an area of intense research in gene therapy. Here, we describe a novel DNA carrier protein termed TG, consisting of the DNA-binding domain of the yeast transcriptional activator GAL4 and human immunodeficiency virus type 1 Tat protein, which can transfer modified naked plasmid DNA into target cells to express foreign genes of interest. The TG protein was expressed in Escherichia coli (E. coli), refolded and purified on an immobilized Ni(2+) affinity chromatography column. SDS-PAGE and Western blotting revealed that the fusion protein was highly expressed with a yield of approximately 275 mg/L. We also constructed the pIRES-UAS-EGFP DNA vector, consisting of upstream activating sequences (UASs) for the specific binding of the DNA-binding protein and the enhanced green fluorescent protein (EGFP) gene. The TG protein could bind specifically to pIRES-UAS-EGFP, forming a complex which could efficiently transfect target cells and result in detectable EGFP protein expression. Thus, these results provide a basis for development of efficient non-viral DNA transfer vectors for further improvements of gene therapy strategies.
Gao
Peng
P
The First Hospital, Bethune Faculty of Medical Sciences of Jilin University, Changchun, PR China.
Li
Xiao
X
Liu
Yanjing
Y
Liu
Yan
Y
Kan
Shifu
S
Jin
Jing
J
Wang
Shuqi
S
Yuan
Changji
C
Jin
Ningyi
N
D026905:Amino Acid Transport Systems; D004268:DNA-Binding Proteins / Q000235:genetics; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D015497:HIV-1 / Q000378:metabolism; D006801:Humans; D010957:Plasmids / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection
D026905:Amino Acid Transport Systems; D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C470089:TAT1 protein, S cerevisiae; D014157:Transcription Factors; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins