CN104672314A - Recombinant archaerhodopsin 4 protein as well as preparation method and application thereof - Google Patents
Recombinant archaerhodopsin 4 protein as well as preparation method and application thereof Download PDFInfo
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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
The invention belongs to gene engineering technology field, be specifically related to a kind of recombinate archaerhodopsin 4 albumen and preparation method thereof and the application in structure biology research.
Background technology
There is a kind of protein family different wavelengths of light to different response in the Nature, be referred to as photosensitive protein.The multiple important role of they performers in the life process of biology, wherein the most famous be exactly in photosynthetic work the keying action that rises.In view of the function that it is special, not only biologist goes all out to study the relation of its structure and function, and material supply section scholar also spares no effort for its light-sensitive characteristic, develops its application in communication and photochromics.In this protein family, bacteria rhodopsin (bacteriorhodopsin, BR) albumen be studied the most deep, technology application and development photosensitive protein the most widely, the so far research history of existing three more than ten years.Bacteria rhodopsin be a kind of polarity that is positioned at addicted to the seven-transmembrane protein on salt bacillus H.salinarum cytolemma, a covalent attachment retinene chromophoric group on its K216 position.When BR there occurs change of configuration by the yellow aldehyde chromophoric group of optical excitation backsight, define a series of light intercycle state with different characteristics absorption peak, thus there is photochromic characteristic.Because BR has the structural performance of seven-transmembrane, similar to important membranin g protein coupled receptor (GPCR) another kind of in organism, be used as the model of the research of GPCR structure biology and Molecular Dynamics Calculation for a long time always.The BR of wild-type is forming stable tripolymer two-dimensional crystal lattice addicted on salt bacillus film, there is the high stability being better than most biomacromolecules, and the light intercycle state to light sensitive and clear and definite characteristic, greatly be better than the photochromic material of existing various chemosynthesis, make it have broad application prospects in optical information storage and optical information processing etc.
Archaerhodopsin (Archaerhodopsin, AR) is the another kind of photosensitive protein similar with bacteria rhodopsin, and it also has proton-pump and light intercycle state.The nineties in last century, find a kind of halophilic bacterium xz515 in China's Zabuye Salt Lake In Tibet, therefrom purifying obtains a kind of membranin equally with light cycle characteristics.By analyzing the aminoacid sequence of this albumen, find the homology with confirmed three kinds of archaerhodopsin albumen with height, therefore called after archaerhodopsin 4 (AR4), and be that China is exclusive.Although AR4 with BR is the same have proton-pump, but proton transfer order is not quite similar with BR, the proton pump transporting mechanism of further investigation AR4 is not only very important to the transmission mechanism of its proton pump of understanding, has very important significance to part still undistinct in explanation BR proton transfer process also tool simultaneously.The more important thing is, the research of AR4 contributes to developing the exclusive optical information of China and stores and optical information processing material.For realizing this goal, the high expression preparation of AR4 different functionalities Residue positions mutant has just become the task of top priority.But, not yet develop the method for the stable expression restructuring AR albumen of any one up to now, let alone obtain corresponding mutant.Have been reported document and relate to the method (Wang, Y.et al.J.Fudan Nat.Sci.2003,42,576-583) that AR4 gene expresses in L33 bacterial strain, but this method existing defects, its experimental result cannot be repeated out.For its defect, the present invention modifies AR4 gene, also carried out part to the leading peptide gene of AR4 to modify simultaneously, final successfully realization is stablized, is given expression to restructuring AR4 albumen efficiently in L33, and constructed multiple mutant, for a series of structure-function research work in later stage are laid a good foundation.
The application of retinene albumen in Optical Information Storage and Processing, key wants to find photosensitive protein to be easy to the two kinds of stable states distinguished.Such as, bacterium visual purple ruddiness circulation M state absorption peak is positioned at 412nm, and ground state absorption peak is then 568nm, and such distribution is just ideal.But be no matter bacteria rhodopsin or the archaerhodopsin of wild-type, their light circulation is very of short duration, and wherein M state is shorter, only has tens milliseconds, is difficult to the Storage and Processing realizing optical information.But existing lot of documents report, some mutant of bacteria rhodopsin greatly can extend the decay of its different intermediate state, the particularly decay of M state, and AR4 this part work corresponding is set up due to expression system delayedly there is not yet report.Therefore, the attenuation characteristic of preparation and research AR4 mutant M state, the optical information exclusive to research and development China stores and optical information processing material is of great immediate significance equally.
Summary of the invention
The object of this invention is to provide a kind of DNA recombinant expression wild-type and saltant type seven-transmembrane protein and recombinant expressed archaerhodopsin 4 albumen and its preparation method and application.
The invention provides a kind of restructuring archaerhodopsin 4 albumen, its its coding nucleotide sequence is as shown in SEQ ID NO.1; Aminoacid sequence is as shown in SEQ ID NO.2; The promoter sequence of regulation and control archaerhodopsin 4 protein expression is as shown in SEQ ID NO.3.
Nucleotide sequence as shown in SEQ ID NO.1:
ATGTTGGAGTTATTGCCAACAGCAGTGATGGACCCGATAGCGCTACAGGCGGGATTCGACCTCCTCGGCGATGGTCGTCCCGAGACACTCTGGTTGGGTATCGGCACGCTACTAATGATCATCGGGACCTTCTATTTCATCGCCCAGGGGTGGGGCGTAACGGATAAGGAGGCCCGTGAGTACTACGCGATCACCATCCTCGTGCCGGGGATCGCGTCGGCCGCGTATCTGGCGATGTTCTTCGGCATCGGTGTGACCGAAGTCGAACTCGCCAGCGGAGCGGTGCTCGACATCTACTACGCCCGGTACGCGGACTGGCTGTTCACGACGCCGCTGCTGCTGCTCGACCTCGCCCTGCTGGCGAAGGTTGACCGCGTCAGCATCGGGACGCTCATCGGCGTCGACGCGCTGATGATCGTCACCGGTCTCATCGGCGCGCTCTCGAAGACGCCGCTCGCGCGGTACACCTGGTGGCTGTTCAGCACGATCGCGTTCCTGTTCGTGCTGTACTACCTCCTGACGAGTCTGCGCAGCGCCGCCGCGCAGCGCTCCGAAGAGGTCCAGAGTACCTTCAACACGCTGACCGCACTGGTCGCCGTCCTCTGGACGGCGTACCCGATCCTGTGGATTGTCGGGACCGAGGGCGCCGGCGTCGTTGGTCTGGGCGTCGAGACCCTGGCGTTCATGGTCCTCGACGTGACCGCGAAGGTCGGATTCGGGTTCGCCCTGCTCCGTAGCCGCGCGATCCTCGGCGAGACCGAGGCCCCCGAGCCGTCGGCCGGCACTTGA
Aminoacid sequence as shown in SEQ ID NO.2:
MLELLPTAVMDPIALQAGFDLLGDGRPETLWLGIGTLLMIIGTFYFIAQGWGVTDKEAREYYAITILVPGIASAAYLAMFFGIGVTEVELASGAVLDIYYARYADWLFTTPLLLLDLALLAKVDRVSIGTLIGVDALMIVTGLIGALSKTPLARYTWWLFSTIAFLFVLYYLLTSLRSAAAQRSEEVQSTFNTLTALVAVLWTAYPILWIVGTEGAGVVG LGVETLAFMVLDVTAKVGFGFALLRSRAILGETEAPEPSAGT
Promoter sequence as shown in SEQ ID NO.3:
CCAGTGAATTCGAGCTCGGTACCCGGGGATCCGAAGTGAAGATGGGGCTCCCGATGGGTGCAACCGTGAAGTCCGTCACGGCTGCGTCACGACAGGAGCCGACCAGCGACACCCAGAAGGTGCGAACGGTTGAGTGCCGCAACGATCACGAGTTTTTCGTGCGCTTCGAGTGGTAACACGCGTGCACGCATCGACTTCACCGCGGGTGTTTCGACGCCAGCCGGCCGTTGAACCAGCAGGCAGCGGGCATTTCACAGCCGCTGTGGCCCACACACTCGGTGGGGTGCGCTATTTTGGTATGGTTTGGAATCCGCGTGTCGGCTCCGTGTCTGACGGTTCATCGGTCTAAATTCCGTCACGAGCGTACCATACTGATTGGGTCGTAGAGTTACACACATATCCTCGTTAGGTACTGTTGC
Restructuring archaerhodopsin 4 albumen of the present invention is archaerhodopsin 4 albumen of transgenation, and the amino acid modified of undergoing mutation is positioned at the aspartic acid of the 97th, belongs to the important residue being positioned at archaerhodopsin april protein subchannel.The generation of this sudden change circulates to the light of archaerhodopsin 4 albumen, proton pump has material impact, and the light intercycle life-span of mutant and recombinant protein of the present invention has under the same conditions and greatly extends compared with wild albumen.Compared with archaerhodopsin 4 albumen extracted with wild-type, the present invention's archaerhodopsin 4 albumen of recombinating has identical proton and extracts and the process of release, and has identical light circulation M state.Because archaerhodopsin 4 albumen that the present invention is recombinant expressed can obtain the high yield sample of milligram quantities level after purifying, therefore can be widely used in structure biology research, such as, can be used for the research of the structure biology such as X-ray crystallography, nuclear magnetic resonance spectroscopy.
Present invention also offers a kind of preparation method of archaerhodopsin 4 albumen of recombinating, it comprises the steps:
(1) gene of restructuring archaerhodopsin 4 albumen is obtained;
With the plasmid pUC19-AR4 template containing AR4 gene, with 5 '-GCCAACAGCAGTGATGGACCCGATAG-3 ' (SEQ ID NO:4) and 5 '-GCCAAGCTTCTAGATCAGTCGCTG-3 ' (SEQ ID NO:5) for primer, DNA fragmentation 1 is obtained by pcr amplification, then with DNA fragmentation 1 for template, with 5 '-ATGTTGGAGTTATTGCCAACAGCAGTG-3 ' (SEQ ID NO:6) and 5 '-GCCAAGCTTCTAGATCAGTCGCTG-3 ' (SEQ ID NO:5) for primer, restructuring archaerhodopsin 4 protein nucleotide sequence is obtained by pcr amplification, as shown in SEQ ID NO.1.
(2) expression vector of restructuring archaerhodopsin 4 albumen is built;
The nucleotide sequence as shown in SEQ ID NO.1 step (1) obtained, splices with the promoter sequence that such as controlling gene shown in SEQ ID NO.3 is expressed.The HindIII restriction enzyme site that spliced sequence is held by the 5 ' BamHI held and 3 ' is connected with pUC19 plasmid, construction recombination plasmid pUC19-bAR; Through BamHI and HindIII double digestion, transfer on pXLNovR plasmid, build and form expression vector pXLNovR-bAR plasmid;
(3) restructuring archaerhodopsin 4 albumen is obtained;
PXLNovR-bAR plasmid is transferred to host cell addicted in salt bacillus (H.salinarum) L33, transform bacterial strain in the medium after cultivating, separation and purification obtains described restructuring archaerhodopsin 4 albumen.
The present invention utilizes genetic engineering technique, and modify 5 ' end of wild AR4 gene, and suddenly change to critical sites, the gene after sudden change proceeds in the bacterial strain not producing retinene albumen, expresses archaerhodopsin mutant.By amplification culture, separation, extraction, purifying obtains not containing sudden change archaerhodopsin 4 albumen of bacterioruberin.
Preparation method of the present invention comprises:
1. the gene overall length 789bp of wild-type AR4, the leading peptide of front 57 alkali yl coding AR4 albumen.The gene of 9 peptides (1-27 bit base sequence: 5'-ATGCTATATGTAGATATGGGTATGGGT-3 ') is held by coding N to replace with in bop gene corresponding part (5'-ATGTTGGAGTTATTGCCAACAGCAGTG-3 '), gene after replacement obtains mRNA through transcribing, its 5 ' end can form loop-stem structure, promotes translation efficiency;
2. AR4 gene and bop gene promoter after modifying splice, called after bAR gene, and pass through HindIII restriction enzyme site that the 5 ' BamHI and 3 ' held holds and pUC19 plasmid is connected to form recombinant plasmid pUC19-bAR, carry out DNA sequencing.Sequencing result shows that its sequence is mated completely with goal gene sequence;
3. with pUC19-bAR plasmid for template, obtain pUC19-bARD97N mutant plasmid with KOD-Plus-Mutagenesis Kit (TOYOBO company) test kit, sequence is errorless through DNA sequencing checking;
4. transferring to pXLNovR plasmid on these two restriction endonuclease sites of BamHI and HindIII by being cloned on pUC19 plasmid and through the bAR gene of sequencing, the pXLNovR-bAR plasmid of gene recombination being transferred to addicted in salt bacillus (H.salinarum) L33;
5. transform bacterial strain and cultivate 12-16 days in containing the substratum of 0.5 μ g/ml Vulkamycin. PA-93, then the garnet bacterium colony that picking color is the darkest 37 DEG C of shaking culture in 10ml is addicted to salt bacillus protein culture medium, when after thalline colour-change, centrifugal detection bacterial sediment color is garnet.Then amplification culture, separation, purifying step by step, finally obtain gene engineering expression not containing restructuring archaerhodopsin 4 albumen of bacterioruberin, i.e. bAR albumen.
Preparation method of the present invention comprises:
1. with pUC19-bAR plasmid for template, obtain pUC19-bARD97N mutant plasmid with KOD-Plus-Mutagenesis Kit (TOYOBO company) test kit, sequence is errorless through DNA sequencing checking;
2. transferring to pXLNovR plasmid on these two restriction endonuclease sites of BamHI and HindIII by being cloned on pUC19 plasmid and through the sudden change bAR gene of sequencing, the pXLNovR-bAR plasmid of gene recombination being transferred to addicted in salt bacillus (H.salinarum) L33;
3. transform bacterial strain and cultivating 12-16 days, the bacterium colony that then picking color is the darkest 37 DEG C of shaking culture in 10ml is addicted to salt bacillus protein culture medium containing in 0.5 μ g/ml Vulkamycin. PA-93, when after thalline colour-change, centrifugal detection bacterial sediment color is garnet.Then amplification culture, separation, purifying step by step, finally obtains not suddenling change archaerhodopsin 4 albumen containing the D97N of bacterioruberin of gene engineering expression.
Wherein, in restructuring archaerhodopsin 4 albumen prepared by the inventive method, the important amino acid that transgenation is modified is positioned at the aspartic acid of the 97th, and this amino acid is the polare Aminosaeren played an important role to proton transfer being positioned at the sub-pump channel of archaerhodopsin april protein.
By preparation method of the present invention can heterogenous expression wild or sudden change restructuring archaerhodopsin 4 albumen, restructuring archaerhodopsin 4 albumen obtained has the function identical with wild-type protein, and this albumen is not containing bacterioruberin, eliminate the impact of bacterioruberin, the protein yield after purifying can reach a milligram rank.Therefore restructuring archaerhodopsin 4 albumen prepared by the inventive method can be used for structure biology research.
Present invention also offers a kind of application of archaerhodopsin 4 albumen in solid-state nuclear magnetic resonance detects of recombinating.
In the present invention's application, in solid-state nuclear magnetic resonance detects, after adding isotope-labeled amino acid cultivate in the substratum of restructuring archaerhodopsin 4 albumen, after separation and purification, obtain isotope-labeled restructuring archaerhodopsin 4 albumen.Preferably, in one embodiment, [U-is obtained after separation and purification
13c
9,
15n]-Leu mark restructuring archaerhodopsin 4 albumen.
The isotope-labeled restructuring AR4 albumen of milligram rank can be obtained by preparation method of the present invention, the solid-state nuclear magnetic resonance spectrogram of gained shows this restructuring archaerhodopsin 4 albumen not containing endophyte red pigment, and there is good space folding, structure biology research can be further used for.
Present invention also offers the application of restructuring archaerhodopsin 4 albumen in Optical Information Storage and Processing.Saltant type archaerhodopsin 4 albumen obtained by preparation method of the present invention, its light circulation M lifetime of intermediate state occurs greatly to extend, and the application of the aspect such as albumen Optical Information Storage and Processing provides possibility for this reason.
The present invention utilizes the method for DNA recombinant expression wild-type and saltant type seven-transmembrane protein archaerhodopsin 4, by genetic engineering technique, suitably modifies AR4 gene and promotor, and proceeds to and carry out protein expression addicted in salt bacillus L33.Protein yield after purified reaches a milligram magnitude, and has the proton pump identical with wild-type AR4 albumen and light circulatory function.Have the amino acid of material impact to carry out sudden change modification to the circulation of the light of archaerhodopsin 4 albumen and proton pump, through to the mutain after recombinant expressed after testing, its light circulation M state life-span can reach the prolongation of the order of magnitude.Mutain of the present invention archaerhodopsin 4 albumen of namely recombinating makes the Optical Information Storage and Processing function of AR4 be optimized, and materials for binding biological is studied, and can be used for the membrane module preparing Optical Information Stovage and Processing.
Accompanying drawing explanation
Fig. 1 represents in embodiment 1, and the bAR gene PCR amplified production of recombination to construct, by the agarose gel electrophoresis result of 1%.
Fig. 2 represents in embodiment 2, and restructuring archaerhodopsin 4 albumen after purifying is detected by the SDSPAGE of 12%, and result and natural archaerhodopsin 4 protein ratio are comparatively.
Fig. 3 represents comparing of wild-type archaerhodopsin 4 albumen that restructuring wild-type archaerhodopsin 4 albumen in embodiment 3 and wild-type are extracted and wild-type BR albumen proton-pump.Wherein, Fig. 3 (A) represents the key gene fragment comprised through the bAR gene of recombination to construct; Fig. 3 (B) represents that bAR gene 5 ' end band has the part identical with bop gene, can form loop-stem structure and promote protein expression; Fig. 3 (C) represents that recombinant expressed archaerhodopsin 4 albumen and natural archaerhodopsin 4 albumen have identical proton and extracts the order with release.
Fig. 4 represents in embodiment 4 M state extinction curve in wild-type archaerhodopsin 4 protein D 97N mutant light circulating reaction of recombinating.
Fig. 5 represents in embodiment 5 M state extinction curve in wild-type archaerhodopsin 4 protein Y 186F mutant light circulating reaction of recombinating.
Fig. 6 represents [U-in embodiment 6
13c
9,
15n]-Leu mark restructuring archaerhodopsin 4 albumen solid-state nuclear magnetic resonance bidimensional single quantum state related experiment result (Fig. 6 (A)) and compare with the superposition of the two quantum state-single quantum state related experiment result (Fig. 6 (B)) of the isotope-labeled BR bidimensional of identical residue.
Embodiment
In conjunction with following specific embodiments and the drawings, the present invention is described in further detail, and protection content of the present invention is not limited to following examples.Under the spirit and scope not deviating from inventive concept, the change that those skilled in the art can expect and advantage are all included in the present invention, and are protection domain with appending claims.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition is as people such as Sambrook, molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number calculate by weight.Implement process of the present invention, condition, reagent, experimental technique etc., except the following content mentioned specially, be universal knowledege and the common practise of this area, the present invention is not particularly limited content.
Embodiment 1:
With wild-type AR4 gene for template, 5 ' end 1-27 bit base of AR4 gene is replaced, concrete grammar is as follows: with the plasmid pUC19-AR4 containing wild-type AR4 gene for template, with 5'-GCCAACAGCAGTGATGGACCCGATAG-3'(SEQ ID NO:4) and 5'-GCCAAGCTTCTAGATCAGTCGCTG-3 ' (SEQ ID NO:5) for primer, DNA fragmentation 1 is obtained by Pfu archaeal dna polymerase pcr amplification, then with DNA fragmentation 1 for template, with 5'-ATGTTGGAGTTATTGCCAACAGCAGTG-3 ' (SEQ ID NO:6) and 5'-GCCAAGCTTCTAGATCAGTCGCTG-3 ' (SEQ ID NO:5) for primer, restructuring archaerhodopsin 4 nucleotide sequence is obtained by Pfu archaeal dna polymerase pcr amplification, as shown in SEQ ID NO.1.
Subsequently, restructuring archaerhodopsin 4 gene is spliced with its promoter sequence of expressing of regulation and control, step is as follows: with the pUC19-bop plasmid containing object promoter sequence for template, with 5'-CGGGATCCGACGTGAAGATGGGG-3 ' (SEQ ID NO:7) and 5'-ACTCCAACATGCAACAGTACCTAAC-3 ' (SEQ ID NO:8) for primer, obtain bop promoter fragment by Pfu archaeal dna polymerase pcr amplification.With archaerhodopsin 4 gene of recombinating for template, with 5'-GTTAGGTACTGTTGCATGTTGGAGT-3 ' (SEQ I D NO:9) and 5'-GCCAAGCTTCTAGATCAGTCGCTG-3 ' (SEQ ID NO:5) for primer, obtain DNA fragmentation 2 by Pfu archaeal dna polymerase pcr amplification.The bop promoter fragment above-mentioned amplification obtained and DNA fragmentation 2 mix, with 5'-CGGGATCCGACGTGAAGATGGGG-3' and 5'-GCCAAGCTTCTAGATCAGTCGCTG-3 ' (SEQ I D NO:5) for primer, undertaken second by Pfu archaeal dna polymerase and take turns PCR, amplification obtains two ends respectively with the gene order of the 1.2kb of BamHI and HindIII restriction site, called after bAR.PCR primer, by the agarose gel electrophoresis of 1%, at the obvious band in 1.2kb place visible, is coincide with expected product size, as shown in Figure 1.Cut glue and reclaim the bAR gene after pcr amplification, after BamHI and HindIII double digestion bAR and pUC19 plasmid, conventionally connect with T4 ligase enzyme, and be transformed into Top10 competent cell.The several clone of random choose on flat board, extracting plasmid, determined the accuracy of goal gene bAR by company's order-checking, sequencing result is consistent with design, by the plasmid called after pUC19-bAR newly built.
Embodiment 2
The transgenation described in the present invention is that the method provided by KOD-Plus-Mutagenesis Kit (TOYOBO company) test kit is realized, concrete steps are as follows: with pUC19-bAR plasmid for template, design two inverse PCR primer, these two primers available linearization template plasmid after pcr amplification, wherein 5 ' end band of a primer has the codon for Mutated residues.With these two primers, carry out pcr amplification with KOD archaeal dna polymerase to template plasmid, obtain the fragment of 3800bp, this fragment is with mutational site.PCR condition is 94 DEG C, 2min, 98 DEG C, 10sec, 68 DEG C, 4min, 15 circulations.In PCR primer, add DpnI restriction enzyme, 37 DEG C process 1 hour, digested plasmid template.Postdigestive PCR primer carries out phosphorylation by phosphokinase, then realizes recirculation with ligase enzyme.Product conversion intestinal bacteria Top10 competent cell after cyclisation, the several clone of random choose on flat board, extracting plasmid, determines the accuracy of mutator gene by company's order-checking.Other details in example 2, the specification sheets that can provide with reference to KOD-Plus-Mutagenesis Kit (TOYOBO company) test kit.
Embodiment 3:
Utilize the method described in embodiment 1,5 ' end of wild AR4 gene is modified, enable the gene after modification in the bacterial strain not producing retinene albumen, express restructuring archaerhodopsin 4 albumen.The gene overall length 789bp of wild-type AR4, the leading peptide of front 57 alkali yl coding AR4 albumen.Hold the gene of 9 peptides to replace with corresponding part in bop gene coding N, the gene after replacement obtains mRNA through transcribing, and its 5 ' end can form loop-stem structure, promotes translation efficiency (Fig. 3 B).AR4 gene after modification and bop gene promoter splice, called after bAR gene (Fig. 3 A), and the HindIII restriction enzyme site held by the 5 ' BamHI and 3 ' held and pUC19 plasmid are connected to form recombinant plasmid pUC19-bAR, carry out DNA sequencing.Sequencing result shows that its sequence is mated completely with goal gene sequence.Transfer to being cloned on pUC19 plasmid and through the bAR gene of sequencing on pXLNovR expression plasmid with these two restriction endonuclease sites of BamHI and HindIII.By protoplast transformation method, the pXLNovR-bAR plasmid of gene recombination is transferred to addicted in salt bacillus (H.salinarum) L33.Transform bacterial strain and cultivate 12-16 days in containing the substratum of 0.5 μ g/ml Vulkamycin. PA-93, then the garnet bacterium colony that picking color is the darkest 37 DEG C of shaking culture in 10ml is addicted to salt bacillus protein culture medium, when after thalline colour-change, centrifugal detection bacterial sediment color is garnet.Then amplification culture step by step, gathers in the crops bacterium after the absorbance at 660nm place is greater than 1.2.4000 × g centrifugal 20 minutes results thalline, the NaCl solution of the product 4M after centrifugal is resuspended, and carries out dialyzed overnight to the NaCl solution of 100mM.Product after dialysis centrifugal 40 minutes through 40000 × g, abandon supernatant, precipitate with deionized water is resuspended.Protein crude extract administration after resuspended is further purified by sucrose gradient centrifugation, finally obtains restructuring archaerhodopsin 4 albumen not containing bacterioruberin of gene engineering expression, called after bAR albumen.SDSPAGE by 12% detects, and display bAR molecular weight of albumen is about 26kD, identical with natural AR4 albumen (Fig. 2).The function of chemical kinetic systems instrument to bAR albumen that room is self-built by experiment detects, result as shown in Figure 3 C, recombinant expressed archaerhodopsin 4 albumen and archaerhodopsin 4 albumen that wild-type is extracted have the order that identical proton extracts and discharges, and extract with the proton of BR-release order is contrary.
Embodiment 4:
Utilize site-directed mutagenesis technique to obtain gene that the 97th aspartic acid (Asp) is mutated into archaerhodopsin 4 albumen of l-asparagine (Asn).With pUC19-bAR plasmid for template, obtain pUC19-bARD97N mutant plasmid with KOD-Plus-Mutagenesis Kit (TOYOBO company) test kit, sequence is errorless through DNA sequencing checking.Transfer to pXLNovR plasmid on these two restriction endonuclease sites of BamHI and HindIII by being cloned on pUC19 plasmid and through the sudden change bAR gene of sequencing, the pXLNovR-bAR plasmid of gene recombination is transferred to addicted in salt bacillus (H.salinarum) L33.Transform bacterial strain and cultivate 12-16 days in containing the substratum of 0.5 μ g/ml Vulkamycin. PA-93, then the bacterium colony that picking color is the darkest 37 DEG C of shaking culture in 10ml is addicted to salt bacillus protein culture medium, when after thalline colour-change, centrifugal detection bacterial sediment color is garnet.Then amplification culture step by step, and carry out albumen sepn and purifying by the method described in embodiment 3, finally obtains not suddenling change archaerhodopsin 4 albumen containing the D97N of bacterioruberin of gene engineering expression.The function of mutain, the chemical kinetic systems instrument that room is self-built by experiment detects, and as shown in Figure 4, the decay of its M state occurs to extend greatly result.
Embodiment 5:
Utilize site-directed mutagenesis technique to obtain gene that the 186th tyrosine (Tyr) is mutated into archaerhodopsin 4 albumen of phenylalanine (Phe).With pUC19-bAR plasmid for template, obtain pUC19-bARY186F mutant plasmid with KOD-PIus-Mutagenesis Kit (TOYOBO company) test kit, sequence is errorless through DNA sequencing checking.Transfer to pXLNovR plasmid on these two restriction endonuclease sites of BamHI and HindIII by being cloned on pUC19 plasmid and through the sudden change bAR gene of sequencing, the pXLNovR-bAR plasmid of gene recombination is transferred to addicted in salt bacillus (H.salinarum) L33.Transform bacterial strain and cultivate 12-16 days in containing the substratum of 0.5 μ g/ml Vulkamycin. PA-93, then the coloured bacterium colony of picking band 37 DEG C of shaking culture in 10ml is addicted to salt bacillus protein culture medium, when after thalline colour-change, centrifugal detection bacterial sediment color is incarnadine.Then amplification culture step by step, and carry out albumen sepn and purifying by the method described in embodiment 3, finally obtains not suddenling change archaerhodopsin 4 albumen containing the Y186F of bacterioruberin of gene engineering expression.The function of mutain, the chemical kinetic systems instrument that room is self-built by experiment detects, and as shown in Figure 5, its M state life-span is slightly longer than wild AR4 albumen for result.
Embodiment 6:
In AR4 culturing process in embodiment 1,2 or 3, respectively the nonisotopic labels leucine in synthetic medium is replaced to
13c,
15the isotope-labeled leucine of N, cultivates restructuring archaerhodopsin 4 albumen and BR albumen.By amplification culture step by step, separation and purification obtains [U-
13c
9,
15n] restructuring archaerhodopsin 4 albumen of-Leu-bAR leucine mark and BR albumen, and carried out two kinds of different bidimensional solid-state nuclear magnetic resonance experiments.
Addicted to salt bacillus synthetic medium composition, as follows:
Amino acid moiety:
Inorganic salt part:
Trace metal ion
Other materials:
Adenylic acid 100mg／L
Uridylic acid 100mg／L
Glycerol 1g／L
Solid-state nuclear magnetic resonance experimental result as shown in Figure 6, owing to comprising in AR4 more than 30 leucines, and is distributed in widely in TM1-TM7, has similar chemical environment.The narrower peak width of the bidimensional single quantum state related experiment result different resonant frequencies of showing illustrates that the self-assembly of restructuring archaerhodopsin 4 in L33 in raw phosphatide is correct, and protein folding is normal and have good space conformation, as shown in Fig. 6 (A).Two quantum state-single quantum state related experiment the result of bidimensional of restructuring archaerhodopsin 4 albumen and BR albumen shows as shown in Fig. 6 (B), the experimental result of what wherein black level line represented is restructuring archaerhodopsin 4 albumen, the experimental result of what Dark grey level line represented is BR albumen, and * indicates is spinning side band.Adopt two quantum state filtering technology only can obtain the structural information of isotopic labeling residue, the transfer of magnetization vector from CO → C α → C β → C γ → C δ clearly can be pointed out out from spectrogram, all spectrum peak positions that restructuring archaerhodopsin 4 albumen and BR albumen chart adding obtain are completely overlapping, illustrate that the phosphatide of restructuring archaerhodopsin 4 albumen and BR albumen is formed consistent, consistent to the modulating action of albumen, not containing bacterioruberin in the interior raw phosphatide of L33.Above solid-state nuclear magnetic resonance experimental result can absolutely prove, restructuring archaerhodopsin 4 albumen adopting the inventive method to prepare can be used for the structure-biological such as nucleus magnetic resonance or X-ray diffraction science study method and resolves Membrane protein conformation and its structure-function relationship of research.
Claims (4)
1. restructuring archaerhodopsin 4 albumen, it is characterized in that, the coding nucleotide sequence of described restructuring archaerhodopsin 4 albumen is as shown in SEQ ID NO.1, and its aminoacid sequence is as shown in SEQ ID NO.2.
2. recombinate the preparation method of archaerhodopsin 4 albumen as claimed in claim 1, it is characterized in that, comprise the steps:
(1) gene of restructuring archaerhodopsin 4 albumen is obtained;
With the plasmid pUC19-AR4 containing AR4 gene for template, with 5'-GCCAACAGCAGTGATGGACCCGATAG-3'(SEQ ID NO:4) and 5'-GCCAAGCTTCTAGATCAGTCGCTG-3 ' (SEQ ID NO:5) for primer, DNA fragmentation 1 is obtained by pcr amplification, then with DNA fragmentation 1 for template, with 5'-ATGTTGGAGTTATTGCCAACAGCAGTG-3 ' (SEQ ID NO:6) and 5'-GCCAAGCTTCTAGATCAGTCGCTG-3 ' (SEQ ID NO:5) for primer, restructuring archaerhodopsin 4 protein nucleotide sequence is obtained by pcr amplification, as shown in SEQ ID NO.1,
(2) expression vector of restructuring archaerhodopsin 4 albumen is built;
The nucleotide sequence as shown in SEQ ID NO.1 step (1) obtained, splices with the promoter sequence that such as controlling gene shown in SEQ ID NO.3 is expressed; The HindIII restriction enzyme site that spliced sequence is held by the 5 ' BamHI held and 3 ' is connected with pUC19 plasmid, construction recombination plasmid pUC19-bAR; Through BamHI and HindIII double digestion, transfer on pXLNovR plasmid, build and form expression vector pXLNovR-bAR plasmid;
(3) restructuring archaerhodopsin 4 albumen is obtained;
PXLNovR-bAR plasmid is transferred to host cell and carries out protein expression addicted in salt bacillus L33, transform bacterial strain in the medium after cultivating, separation and purification obtains described restructuring archaerhodopsin 4 albumen.
3. preparation method as claimed in claim 2, is characterized in that, restructuring archaerhodopsin 4 albumen that described preparation method obtains is not containing bacterioruberin.
4. the application of restructuring archaerhodopsin 4 albumen in solid-state nuclear magnetic resonance detects as claimed in claim 1, it is characterized in that, cultivate by adding isotope-labeled amino acid in the substratum of restructuring archaerhodopsin 4 albumen, separation and purification obtains isotope-labeled restructuring archaerhodopsin 4 albumen.
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