The Study Of Construction And Purification Of Thioredoxin Fusion Protein Expression Vetor

Molecular biological techniques have been widely used in many research fileds of modern life sciences, as well as environment health. Because of the difficulty of obtaining the aim protein or polypeptide, to some extent, the development of environment health is obstacled. Now protein expression technique is necessary in the studies of environment health, such as environmental genomics, mechanisms of interactions between cell and pollutants. Among the techniques, fusion protein ex-prerssion received much attentation because of its high expression level and ease of purification in E. coli, yeast and other host. Most importantly , fusion proteins still have compatible activity and function of the specific nature protein. Therefore, fusion protein expression technique should be widely used for obtaining the aim products in many studies.Glutathoine S -transferase (GST) and thioredoxin (Trx) are the tags often used in fusion protein expression. The purification of GST fusion protein has to use GST affinity chromatography. However, the appliacation of this method is limited due to the expense of the affinity media and the difficulty of removing the GST tag from fusion protein, which needs expensive specific proteinase.Trx is a kind of chaperons which mediate the correct folding of proteins. As we all know that most proteins will be denatured afterheating, however, Trx is stable even at temperature as high as over 90 , so Trx fusion protein could be purified easily from background proteins through heating. However, the use of this novel expression vector is obstacled due to the low expression level and expensive enzymatic purification process. Therefore, it is necessary to modify the commercial vectors to realize the high level expression and make the purification process easier and cheaper. CNBr is a kind of cheap chemical often used in specific cleavage of Met - X ( X means any a-mino acid) in proteins or peptides. But the 38th ammo acid of Trx is Met, when CNBr is used to cleave the fusion tag, it will make the purification complicated. As the thioredoxin molecule is small and its struture is simple, modification of the gene would be easy. The purposes of this study are to construct a novel Trx fusion expression vector using site - mutation technique and then test the methods for expression and purification of the Trx fusion protein using the nomatod an-tithrombotic peptide (ATP) model.MATERIALS AND METHODSMaterials: E. coli BL21 (DE3) , DH5a and polyclonal antibody against fusion protein GST - ATP were all from, the Institute of Vir -gology, Chinese Academy of Preventive Medicine. All primers used in this paper were synthesized by Shanghai ShengGong Bio - tech Company. Other materials were bought from different factories or companies domestic or abroad.Expressing - vector pEilla containing T7 RNA polymerase pro-motor was from Novagen Company. Plasmid pThioHisA which owned the gene Trx was bought from Invitrogen Company. pCDNA II - MAPcontained the full length cDNA of ATP and cloning vector pCDNA II were obtained from the Institute of Virgology, Chinese Academy of Preventive Medicine.Methods: Site mutation of Trx gene was conducted by using PCR method to change the Met38 (ATG) to Ala (GCG). The PCR products was recovered and cloned into pCDNA II and sequenced to confirm the mutation. Then the full length ATP gene was also amplified with PCR. The target fragment was recovered and inserted into pCD-NA II cloning vector and sequenced. The sequencing confirmed muta-tant of Trx, which was named Trxm, was then inserted into the expression vector pETlla after digested with Nde I and BamH I and recovered. The resulted plasmid was named pET - Trx, which could be used as a fusion expression vetor in E. coli. The ATP gene in pCDNA II - ATP was obtained after cleaved with Xho I and BamH I and was inserted into the Xho I - Bam HI site of pET - Trx. The resulted vector which could express Trx - ATP fusion protein was named pET -Trx/ATP. The plasmid was then transformed into E. coli BL21 ( DE3 ) to tes...