[Separate expression of ABCA1 and ligation mediated by Ssp DnaE intein in Escherichia coli BL21 (DE3)].
OBJECTIVE
By exploring Ssp DnaE intein-catalyzed protein trans-splicing we investigated the ligation of expression product of ATP-binding cassette transporter A1 (ABCA1) gene in E. coli.


METHODS
The ABCA1 cDNA was broken into two halves of N-part and C-part before Cys978 codon which meets the splicing required conserved residue, and then fused to 123 and 36 amino acid-containing N terminal and C terminal coding sequences of Ssp DnaE intein naturally occurring trans-splicing intein respectively. These two fusion genes were constructed into a prokaryotic expression vector pET-28a (+). After transformation into E. coli BL21 (DE3) cells followed by induction the expression of recombinant proteins and the ligation of ABCA1 were observed.


RESULTS
Through IPTG induction for expression of recombinant protein it displayed an obvious protein band as predicted size of ABCA1 on SDS-PAGE gel. Western blotting using His-Tag specific antibody confirmed that this protein band is trans-spliced ABCA1.


CONCLUSIONS
The data demonstrated that Ssp DnaE intein can efficiently catalyze the ligation of ABCA1 providing an evidence for our ongoing study on ABCA1 gene transfer by a dual AAV vector system to circumvent AAV volume limitation in gene therapy of Tangier disease which resulted from ABCA1 gene mutations.
Zhu
Fuxiang
F
Life Science College, Ludong University, Yantai 264025, China. fuxiangmail@163.com
Miao
Jing
J
Qu
Huige
H
Chi
Xiaoyan
X
D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000378:metabolism; D004258:DNA Polymerase III / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D047668:Inteins; D019154:Protein Splicing
C577836:ABCA1 protein, human; D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters; D029968:Escherichia coli Proteins; C072071:SspA protein, E coli; C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III