Cloning, Expression, And Purifacation Of Torsin A Encoded By Human DYT1 Gene

Dystonia describes a neurologic condition characterized by involuntary, sustained muscle contractions affecting one or more sites of the body; "torsion" refers to the twisting nature of body movements observed in dystonia. Dystonia has been classified as primary (dystonia as the sole or major symptom) or secondary (a symptom of another disorder), and by age of onset, muscle groups affected, and mode of inheritance.Ozelius et al. found a 3-bp deletion in the DYT1 gene in all affected and obligate carrier individuals with chromosome 9-linked primary dystonia, regardless of ethnic background and surrounding haplotype. The deletion resulted in loss of 1 of a pair of glutamic acid residues; GAG was deleted from a GAGGAG sequence that is conserved in all human, rat, and mouse torsinA and torsinB transcripts, suggesting that it is part of a functional domain. From analysis of 3 new single-basepair polymorphisms in a 5-kb region surrounding the GAG deletion, it was concluded that the same mutation must have arisen more than once. The finding of the same 3-bp mutation in heterozygous state in most cases of typical early-onset dystonia is comparable to the few examples of the same recurrent mutation causing other dominantly inherited conditions. These include the FGFR3 mutation responsible for almost all cases of achondroplasia and the loss of a positively charged arginine in the fourth transmembrane helix of the alpha-1 subunit of the L-type voltage-sensitive calcium channel, which Ozelius et al. noted, was the only type of mutation found thus far to cause hypokalemic periodic paralysis. In thesecases, as well as in the case of the CAG expansions in the coding regions of a number of genes causing neurodegenerative diseases, the same mutations occurred repeatedly as independent events, whereas other mutations in the same gene cause a different syndrome, have no phenotype, or are incompatible with life.The study is to construct the prokaryotic expression vector of human torsion A protein and obtain its expression and purification in E coli. The biological information of torsion A protein is analysed by using Blast analysis, transmembrane analysis, signal peptide prediction, basic physical & chemical character analysis, and predictions of both secondary and three dimentinal constructions. A pair of primers is designed spanning the complete coding sequence of DYT1 without the coding sequence of its signal peptide. A polymerase chain reaction(PCR) is performed using this pair of primers and the human liver cDNA library. And a cDNA fragment of 939 bp, which includes the complete coding sequence of mature DYT1 protein, is amplified and retracted by 1% gelose gel electrophoresis. Then a reconstructedplasmid of DYT1 coding region cDNA------pMD 18-T-DYT1, is constructed byinserting the cDNA fragment into pMD 18-T vector and selecting the sense clones by PCR and restriction enzyme analysis. The sequencing result is consistente with that of reported. The plasmid is digested with Bam H I and Xho I in 37 ℃for 3 h, and the 939bp fragment is purified and cloned into the prokaryotic expression vectors pET28(+) and pGEX-6P-l. The receptor bacteria JM109 is transformed, the plasmid is extracted from JM109 and then transformed into the competent cell of BL21(DE3) of E.coli. Postive clone is identified and the prokaryotic expression vector of torsion A is then constructd successfully. The constructed E coli. is inoculated into the antibiotic culture medium .The cultured bacteria liquid is inoculated in 250 mL of LB liquid culture medium in the ratio of 1:100 in the next day,and then cultured vibratingly in 37℃. The cultured bacteria are induced by IPTG when the absorbency of 600nm gets about 0.6. The bacteria are gathered, thesedimentation and the clear liquid are obtained by ultrasonic fragmentizing and centrifuging. The expression and the solubility of the protein are identified by SDS-polyacrylamine gel electrophoresis analysis. A 40 kD DYT1-6 His fusion protein and a 66 kD DYT1-GST fusion protein are highly expressed in pET28a and in pG...