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PROJECT
EXPERIMENTS
Making functional long chain protein
In our last study, our long protein lost its function. As the reason, we have the idea that the protein lost its holding because it was made be long. For the reason of this problem, we have the idea that being long protein chain destructs its holding. So in this year, we aim to synthesize functional long chain proteins by designing them including some linkers.
The requirements for amino acids that con be the linkers are less of steric effects and strong in hydropathy. Glycine and serine fulfill this requirements. Glycine’ side chain has almost no steric effects. On the other hand, serine has some steric effects but it has powerful hydropathy because of its hydroxyl side chain. According to やべっち, because histidine has quite strong hydropathy, though it has big steric effects, it is possible that histidine can work as linker.
Amino acid chains synthesized from circular messenger RNA are constructed from repetition in amino acid of target protein, amino acid from ribozyme and amino acid from RBS. In this experiment, we inserted linker sequences into inside of both splicing sites. So, protein chains synthesized from circular messenger RNA that include linker sequences repeat linker sequence, amino acid from RBS, target protein, linker sequence and amino acid from ribozyme.
Generally speaking, length of linker is required of one-forth of diameter of target protein. In the case of RFP, that condition is surely cleared by six amino acid, but it is unknown which linkers are appropriate in order to synthesize poly RFP protein. Therefore, we examine some types of amino acids and length of linker and choice the best linker in this experiment.
developing the efficiency of circularization
Figure: prediction of secondary structure of td gene intron and around splicing site
Because the complementary sequence designed inside the splicing site is included circular mRNA, its sequence is translated into amino acids. So we designed this sequence as complementarity and efficiency.
Because the base sequence of ① and ③ are almost complementary, it expects that combining these base sequences make the cyclization more efficient.
We made 9 kinds of devices by combining 7 kinds of linkers. Please look at a below list.
We made these devices and analyzed the long chain protein and its functionality.
And we also analyzed efficiency of cyclization about the devices of using linker ①-③,③-①.
To raise the efficiency of cyclization, we designed the complementary sequences at outside or inside of the splicing site. The sequences that designed inside the splicing site are linker ①-③,③-①. The sequence designed outside the splicing site is the sequences td gene originally has and we cloned the sequence that includes these sequences and make them parts.
Figure :cloning part that is inserted complementary sequences outside both splicing sites.
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