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Revision as of 06:42, 8 October 2014
1.Fusion protein and linkers
Fusion protein was a genetic expression product, which was composed by two or more head-to-tail ligation coding region that were controlled by the same regulatory sequence. The design of linker played an important part in the fusion protein with connecting peptides.
The advantages of fusion enzymes lie in that it is able to integrate a variety of active catalytic enzyme in a protein. In the process of a large number of sequential enzyme catalysis, the product of an enzyme (intermediate) is the substrate of next step of enzyme catalytic reaction, and these enzymes are often in a free state especially in plant cells, so the distance between the enzyme molecule is difficult to control, which restricts its catalytic efficiency. Our solution is to connect multiple lysosomal enzymes through the fusion protein technology and put them in the sequence of the catalytic reaction to adjust the space structure to the effect, thus we can control of reaction efficiency.
Peptide linkers are used to connect protein domains. For this connection it is important that the linker itself has no influence on the connected parts. Therefore the sequence of the linker is designed for amino acids which do not interact with other amino acid residues. The amino acids glycine and serine are zwitterionic and hydrophile, these properties make them a good choice for the repetitive sequence of the linker.
The length of the linker is important to guarantee the independent function of two connected parts. When the linker is too short there might be a sterical interference between the parts and if it is too long, the construct can be instable. Also, it is important that the linker has a certain flexibility.
Based on these rules we designed our linkers to make the fusion gene crtEBI and crtYZW. The sequence of the linker is: GGTAGCGGCTCCGGAAGCGGTTCCGGCAGC.
