[Separate expression of ABCA1 and ligation mediated by Ssp DnaE intein in Escherichia coli BL21 (DE3)].

Abstract

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.

CONCLUSION:

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.

• Authors: Fuxiang Zhu, Jing Miao, Huige Qu, Xiaoyan Chi
• Journal: Wei sheng wu xue bao = Acta microbiologica Sinica (Wei Sheng Wu Xue Bao) Vol. 49 Issue 12 Pg. 1601-6 (Dec 2009) ISSN: 0001-6209 [Print] China
• PMID: 20222445 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• ABCA1 protein, human
• ATP Binding Cassette Transporter 1
• ATP-Binding Cassette Transporters
• Escherichia coli Proteins
• SspA protein, E coli
• DNA polymerase III, alpha subunit
• DNA Polymerase III

Topics

• ATP Binding Cassette Transporter 1
• ATP-Binding Cassette Transporters (genetics, metabolism)
• DNA Polymerase III (genetics, metabolism)
• Escherichia coli (genetics, metabolism)
• Escherichia coli Proteins (genetics, metabolism)
• Humans
• Inteins
• Protein Splicing