Isolated aQdecay photoprotein and use thereof
A light-emitting protein and bioluminescence technology, which can be used in recombinant DNA technology, introduction of foreign genetic material using vectors, peptide sources, etc., and can solve the problem of unclear importance of fluorescence.
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Embodiment 1
[0162] To prepare the variants, mutants were inserted at position 132 (truncating position 132 of aequorin; GenBank #AAA27716) by means of molecular biological methods. For this purpose, the "Quick change" method provided by the company Stragene (USA) was used. The primers used were (SEQ ID NO: 3) and (SEQ ID NO: 4). The cDNA was inserted into the NdeI / XhoI cleavage site of the vector pET22b (Novagen). Vehicle is indicated as pET22b-AQdecay.
[0163] figure 1 The plasmid map of the vector pET22b-AQdecay is shown.
Embodiment 2
[0165] Plasmid pcDNA3.1(+) provided by Clontech was used as a vehicle for the preparation of the constructs described below. A derivative of the vector is denoted as pcDNA3-AQdecay. The vector pcDNA3-AQdecay was used to express AQdecay in eukaryotic systems.
[0166] figure 2 The plasmid map of the vector pcDNA3-AQdecay is shown.
Embodiment 3
[0168] bacterial expression
[0169] Bacterial expression in Escherichia coli was achieved by transforming the bacteria with the expression plasmid pET22b-AQdecay. Transformed bacteria were grown in LB medium for 3 hours at 37°C, and their expression was induced according to the manufacturer's (Novagen) instructions. Induced bacteria were collected by centrifugation, resuspended in 50 mM Tris / HCl (pH 9.0) + 5 mM EDTA and disrupted by sonication. Then, the resulting lysate was centrifuged at 13000 rpm (16000 rcf) for 15 minutes, and the supernatant was removed. In the dark, supernatant (1:5; 1:10; 1:20 and 1 :50 dilution) for 3 hours. Bioluminescence was measured in a luminometer immediately after addition of 5 mM calcium chloride. The measurement accumulation time was 40 seconds.
[0170] Figure 4 Kinetics of AQdecay bioluminescence measurements in bacteria are shown.
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