Team:CSMU NCHU Taiwan/Design
Design
Thioredoxin fusion system
In our project, we used E. coli to express two heterogeneous enzymes from mycobacterium smegmatis. However, we didn’t know whether they were toxic to E. coli and whether they would become inclusion bodies because of insolubility when E. coli produced them. Therefore, we found the thioredoxin fusion proteins system. When the gene of thioredoxin in E. coli (TrxA) were co-expressed, the target proteins were inserted into the active-site loop of thioredoxin, and therefore the fusion proteins can be more soluble[1].
In addition, between the thioredoxin domain and the target gene domain, we designed some linkers to ligase two domains. The first part, which could be translated to the peptide sequence, “DDDDK”, was designed as the cleavage site of enterokinase. There were some restriction sites in the second part. The third one, which could be translated to glycine-glycine, was a flexible linker[2].
Fig. 1: BBa_K2382004 showed the gene design of the Thioredoxin fusion system construction.
Aflatoxin-degrading enzyme: F420-dependent reductase group A
F420-dependent reductases (FDR) can be divided into two classes (A and B) and be found in some species of bacteria[3]. FDR-A enzymes, has up to 100 times more activity than the other. In this class, MSMEG5998 has the best specific activity to AFB1 (10350 nmol/min/mol enzyme) and AFG1 (103210 nmol/min/mol enzyme). Therefore, we looked for the coding sequence of MSMEG5998, which was registered in NCBI in mycobacterium smegmatis and put the sequence of thioredoxin before it to form a fusion protein. For the purpose of purification through nickel-resin column, we added a 18-bp sequence which can code 6 histidines. In addition, we chose T7 promoter which contains lac operator to express this protein because it can be induced by IPTG. For the terminator, we chose BBa_B0015 because it was commonly used in E. coli. The gene design are shown in Fig. 2.
Fig. 2: BBa_K2382006 showed the gene design of the Thioredoxin-MSMEG5998 fusion protein construction.
The activator ...
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