Post-translational ligation of split CFTR severed before TMD2 and its chloride channel function.
- Author:
Fuxiang ZHU
1
;
Xiandi GONG
;
Zelong LIU
;
Shude YANG
;
Huige QU
;
Xiaoyan CHI
Author Information
1. College of Life Science, Ludong University, Yantai 264025, China. fuxiangmail@163.com
- Publication Type:Journal Article
- MeSH:
Animals;
Cell Line;
Chloride Channels;
physiology;
Cricetinae;
Cystic Fibrosis Transmembrane Conductance Regulator;
biosynthesis;
genetics;
Dependovirus;
genetics;
Genetic Vectors;
Humans;
Membrane Potentials;
genetics;
Protein Processing, Post-Translational;
Protein Splicing
- From:
Chinese Journal of Biotechnology
2010;26(12):1710-1716
- CountryChina
- Language:Chinese
-
Abstract:
Mutations of cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to cystic fibrosis, an autosomal recessive genetic disorder affecting a number of organs including the lung airways, pancreas and sweat glands. In order to investigate the post-translational ligation of CFTR with reconstructed functional chloride ion channel and the split Ssp DnaB intein-mediated protein trans-splicing was explored to co-deliver CFTR gene into eukaryotic cells with two vectors. The human CFTR cDNA was split after Glu838 codon before the second transmembrane dome (TMD2) into two halves of N- and C-parts and fused with the coding sequences of split Ssp DnaB intein. Pair of eukaryotic expression vectors pEGFP-NInt and pEYFP-IntC were constructed by inserting them into the vectors pEGFP-N1 and pEYFP-N1 respectively. The transient expression was carried out for observing the ligation of CFTR by Western blotting and recording the chloride current by patch clamps when cotransfection of the pair of vectors into baby hamster kidney (BHK) cells. The results showed that an obvious protein band proven to be ligated intact CFTR can be seen and a higher chloride current and activity of chloride channel were recorded after cotransfection. These data demonstrated that split Ssp DnaB intein could be used as a strategy in delivering CFTR gene by two vectors providing evidence for application of dual adeno-associated virus (AAV) vectors to overcome the limitation of packaging size in cystic fibrosis gene therapy.
