Phycocyanin and phycoerythrin beta subunits fluorescent protein combined with phycoerythrobilin PEB and application thereof
A technology of phycoerythrin and phycoerythrin, which is applied to the phycoerythrin β subunit fluorescent protein combined with phycoerythrin PEB and its application field, can solve the problem of high background, complex technical procedures, fluorescent immunology Technical quantitative determination is difficult and other problems, to achieve the effect of good sensitivity, easy purification, high fluorescence efficiency
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Embodiment 1
[0057] The amino acid sequence of the protein is shown in Sequence 1. The phycoerythrin beta subunit encoding gene is cloned into an expression plasmid, and the phycoerythrin beta subunit is expressed, and its N-terminus has a His-tag mark, which is not only beneficial to its Purification also helps to increase its solubility. Phycoerythrin is bound to the cysteine residue at position 130 (equivalent to position 82 of the original phycoerythrocyanin beta subunit) through a thioether bond. Its spectrum is as figure 2 As shown, the absorption peak is 546nm, and the fluorescence emission peak is 566nm.
Embodiment 2
[0059] The amino acid sequence of the protein is shown in Sequence 2. The gene encoding the phycoerythrin beta subunit is cloned into an expression plasmid, and mutated by genetic engineering methods to obtain a phycoerythrin beta subunit mutant with an N-terminal band There is a His-tag mark, which not only facilitates its purification, but also helps to improve its solubility. Phycoerythrin is bound to the cysteine residue at position 130 (equivalent to position 82 of the original phycoerythrocyanin beta subunit) through a thioether bond. Its spectrum is as image 3 As shown, the absorption peak is 546nm, and the fluorescence emission peak is 566nm.
Embodiment 3
[0061] The amino acid sequence of the protein is shown in Sequence 3. The streptavidin encoding gene and the phycoerythrin beta subunit encoding gene were spliced and cloned into the expression plasmid, and the streptavidin and phycoerythrin beta subunit were expressed. The fusion protein of the phycoerythrin beta subunit can be directly marked by streptavidin; and the N-terminal has a His-tag tag, which not only facilitates its purification, but also helps to improve its solubility. Phycoerythrobilin is bound to the cysteine residue at position 258 (equivalent to position 82 of the original phycoerythrocyanin beta subunit) through a thioether bond. Its spectrum is as Figure 4 As shown, the absorption peak is 546nm, and the fluorescence emission peak is 566nm.
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