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Recombinant archaerhodopsin 4 protein as well as preparation method and application thereof

A violin and protein technology, applied in the field of genetic engineering, can solve the problems of prolonged attenuation, short light cycle, difficult to realize optical information storage and processing, etc., and achieve the effect of good spatial folding

Active Publication Date: 2015-06-03
EAST CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, whether it is wild-type bacteriorhodopsin or ancient rhodopsin, their light cycle is very short, and the M state is even shorter, only a dozen milliseconds, making it difficult to store and process light information
However, there have been a large number of literature reports that some mutants of bacteriorhodopsin can greatly prolong the attenuation of its different intermediate states, especially the attenuation of the M state, and the corresponding part of the work of AR4 has not been reported due to the lag in the establishment of the expression system.

Method used

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  • Recombinant archaerhodopsin 4 protein as well as preparation method and application thereof
  • Recombinant archaerhodopsin 4 protein as well as preparation method and application thereof
  • Recombinant archaerhodopsin 4 protein as well as preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0047] Using the wild-type AR4 gene as a template, replace the 1-27 bases at the 5' end of the AR4 gene. (SEQ ID NO: 4) and 5'-GCCAAGCTTCTAGATCAGTCGCTG-3' (SEQ ID NO: 5) as primers, the DNA fragment 1 was obtained by PCR amplification with Pfu DNA polymerase, and then the DNA fragment 1 was used as a template, and the 5'- ATGTTGGAGTTATTGCCAACAGCAGTG-3' (SEQ ID NO: 6) and 5'-GCCAAGCTTCTAGATCAGTCGCTG-3' (SEQ ID NO: 5) were used as primers, and the recombinant ancient rhodopsin 4 nucleotide sequence was obtained by PCR amplification with Pfu DNA polymerase, such as SEQ ID NO.1 is shown.

[0048]Subsequently, the recombinant ancient rhodopsin 4 gene was spliced ​​with the promoter sequence that regulates its expression. The steps are as follows: use the pUC19-bop plasmid containing the target promoter sequence as a template, and use 5'-CGGGATCCGACGTGAAGATGGGG-3' (SEQ ID NO: 7) and 5'-ACTCCAACATGCAACAGTACCTAAC-3' (SEQ ID NO: 8) were used as primers, and the bop promoter fragment w...

Embodiment 2

[0050] The gene mutation described in the present invention is realized by the method provided by the KOD-Plus-Mutagenesis Kit (TOYOBO company), and the specific steps are as follows: using the pUC19-bAR plasmid as a template, two reverse PCR primers are designed. The primers can linearize the template plasmid after PCR amplification, and one of the primers has a codon for the residue to be mutated at the 5' end. Using these two primers, use KOD DNA polymerase to perform PCR amplification on the template plasmid to obtain a 3800bp fragment, which already has a mutation site. The PCR conditions were 94°C, 2min, 98°C, 10sec, 68°C, 4min, 15 cycles. Add DpnI restriction endonuclease to the PCR product, treat at 37°C for 1 hour, and digest the plasmid template. The digested PCR product is phosphorylated by phosphokinase and then self-circularized by ligase. The circularized product was transformed into Escherichia coli Top10 competent cells, several clones were randomly selected ...

Embodiment 3

[0052] Using the method described in Example 1, the 5' end of the wild AR4 gene was modified so that the modified gene could express the recombinant rhodopsin 4 protein in a strain that does not produce retinal protein. The total gene length of wild-type AR4 is 789bp, and the first 57 bases encode the leader peptide of AR4 protein. The gene encoding the nine peptides at the N-terminal is replaced with the corresponding part of the bop gene, and the replaced gene is transcribed to obtain mRNA, and its 5' end can form a stem-loop structure to promote translation efficiency ( image 3 B). The modified AR4 gene was spliced ​​with the bop gene promoter, named bAR gene ( image 3 A), and connect the pUC19 plasmid through the BamHI at the 5' end and the HindIII restriction site at the 3' end to form a recombinant plasmid pUC19-bAR, and perform DNA sequencing. Sequencing results showed that its sequence completely matched the sequence of the target gene. The bAR gene cloned on the ...

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Abstract

The invention provides a recombinant archaerhodopsin 4 protein. An encoding nucleotide sequence is shown in SEQ ID NO.1, and an amino acid sequence is shown in SEQ ID NO.2. The invention also provides a preparation method of the recombinant archaerhodopsin 4 protein. By utilizing a gene engineering method, an AR4 gene is appropriately modified, and converted into halobacterium L33 for performing protein expression. The invention also provides application of the recombinant archaerhodopsin 4 protein in structural biology research.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a recombinant ancient rhodopsin 4 protein, a preparation method thereof and an application in structural biology research. Background technique [0002] In nature, there is a family of proteins that respond differently to light of different wavelengths, called photosensitive proteins. They play a variety of important roles in the life process of organisms, the most famous of which is the key role played in photosynthesis. In view of its special function, not only biologists devote themselves to studying the relationship between its structure and function, but material scientists also spare no effort to develop its application in communication and photosensitive materials for its light-sensitive properties. In this protein family, bacterial rhodopsin (bacteriorhodopsin, BR) protein is the light-sensitive protein that has been studied the most deeply and ha...

Claims

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Application Information

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IPC IPC(8): C07K14/215C12N15/31C12N15/74G01N24/08C12R1/01
CPCC07K14/215G01N24/087
Inventor 赵欣曹振
Owner EAST CHINA NORMAL UNIVERSITY
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