Part:BBa_K5040000
Zif268-rlinker- m-hTPH1
The fusion protein of m-hTPH1 and Zif268 binding domain with 36 bp rigid linker. m-hTPH1 is truncated form of TPH genes, TPH1 mutant with a deletion of first 99 N-terminal and last 24 C-terminal amino acids. Zif268 is a naturally occurring 3-finger ZF. It binds the 9 bp sequence 5'-GCG TGG GCG-3'
Experimental result
After confirming the cloning, we induced the Zif268-rlinker-m-hTPH1 fusion protein expression by treating IPTG at 37°C in different induction time and IPTG concentration.
Fig. The Coomassie Blue staining of SDS-PAGE analysis of the Zif268-rlinker-m-hTPH1 fusion protein at 0, 4, and 8 hours of IPTG induction. Sup: supernatant; P: pellet
As presented above, we discovered that the individual expression of the Zif268-rlinker-m-hTPH1 fusion protein was unsuccessful; therefore, we extended induction time and increased IPTG concentration.
Fig. The Coomassie Blue staining of SDS-PAGE analysis of the Zif268-rlinker-m-hTPH1 fusion protein at 0, 12, and 24 hours of IPTG induction. Sup: supernatant; P: pellet
Fig. The Coomassie Blue staining of SDS-PAGE analysis of the Zif268-rlinker-m-hTPH1 fusion protein induced with IPTG concentrations of 0.2 mM, 0.4 mM, and 0.8 mM at 8 hours.
However, we also found that co-expressing Zif268-rlinker-m-hTPH1, PBSII-rlinker-hPCBD1, and ZFa-hQDPR resulted in successful expression of Zif268-rlinker-m-hTPH1.
Fig. The Coomassie Blue staining of SDS-PAGE analysis of co-expressing Zif268-rlinker-m-hTPH1, PBSII-rlinker-hPCBD1, and ZFa-hQDPR fusion protein at 0, 4, and 8 hours of IPTG induction. Sup: supernatant; P: pellet
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 338
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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