Recombinant contained-double chromophore photoreceptor protein archaerhodopsin 4 and construction method and application thereof
A receptor protein, dual-color technology, applied in the field of genetic engineering, can solve the problems of low expression efficiency, inability to express and contain bacteriocin photoreceptor protein, waste of resources, etc.
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Embodiment 1
[0028] Example 1: Using PCR to construct the expression vector pMPK66-aR4 of archaeordopsin 4 protein
[0029] Using the plasmid pUC19-aR4 containing the aR4 gene as a template, using 5'-CGGGATCCGACGTGAAGATGGGG-3'(SEQ ID NO:3) and 5'-CTCAAGCTTGGGCCCTGCTAGTCGTCGGG-3'(SEQ ID NO:4) as primers, through chain polymerase Reaction (Polymerase Chain Reaction, PCR) amplifies the aR4 fragment that DNA fragment 1 promptly end contains restriction endonuclease PspOMI site (5'-G^GGCCC-3'), as figure 1 shown. And utilize restriction endonuclease MIUI and PspOMI to connect it on the pBA2 (SEQ ID NO:5) plasmid, construct the expression vector pMPK66-aR4 plasmid of ancient rhodopsin 4 protein, flow process is as follows figure 2 As shown, the main steps are as follows:
[0030] 1) Using the existing pUC19-aR4 plasmid in the laboratory as a template, use the upstream primer and downstream primer (containing restriction site) synthesized by Shanghai Boshang Biological Co., Ltd. to amplify the...
Embodiment 2
[0046] Using the method described in Example 1, after successfully preparing pMPK66-aR4, the expression plasmid was transformed into Halobacterium MPK409 by protoplast transformation. Transformed strains were cultured in PM solid medium containing 5-fluoroorotic acid (5-Fluoroorotic Acid, 5-FOA) and lovastatin (Mevinolin, Mev) for 12-16 days, and then picked the darkest dark red colony Shake culture in 10ml peptone medium at 37°C, and add 5-FOA with a final concentration of 0.25mg / ml and Mev with a final concentration of 4μg / ml. When the color of the bacteria changes, centrifuge to detect that the color of the precipitate of the bacteria is dark red. It is considered that a protein expression strain of aR4 was obtained.
Embodiment 3
[0048] Utilize the expression bacterial strain that the method described in embodiment 2 obtains, amplify culture step by step, the substratum that pre-cultivation uses is CM substratum, transfers in PM substratum (CM and PM substratum) after amplifying to OD value 0.4 See the composition below), continue to expand the culture at 37°C with light and shaking, and harvest the bacteria when the OD value at 660nm is greater than 1.2. The cells were harvested by centrifugation at 4000×g for 20 minutes, and the centrifuged product was resuspended with 4M NaCl solution, and dialyzed overnight against 100 mM NaCl solution. The dialyzed product was centrifuged at 40,000×g for 40 minutes, the supernatant was discarded, and the precipitate was resuspended with deionized water. The resuspended protein crude extract was further purified by sucrose gradient centrifugation to finally obtain the rhodopsin 4 protein containing bacteriocin, which was detected by 12% SDS-PAGE, showing that the m...
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