CN104672314B - One kind restructuring 4 albumen of archaerhodopsin and its preparation method and application - Google Patents
One kind restructuring 4 albumen of archaerhodopsin and its preparation method and application Download PDFInfo
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- CN104672314B CN104672314B CN201310630099.9A CN201310630099A CN104672314B CN 104672314 B CN104672314 B CN 104672314B CN 201310630099 A CN201310630099 A CN 201310630099A CN 104672314 B CN104672314 B CN 104672314B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/215—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Halobacteriaceae (F)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N24/00—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects
- G01N24/08—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects by using nuclear magnetic resonance
- G01N24/087—Structure determination of a chemical compound, e.g. of a biomolecule such as a protein
Abstract
The present invention provides one kind to recombinate 4 albumen of archaerhodopsin, its coding nucleotide sequence is as shown in SEQ ID NO.1, its amino acid sequence is as shown in SEQ ID NO.2.Present invention also offers the preparation method of restructuring 4 albumen of archaerhodopsin, AR4 genes are suitably modified using gene engineering method, and be transferred in thermophilic salt bacillus L33 and carry out protein expression.Present invention also offers application of restructuring 4 albumen of archaerhodopsin in structure biology research.
Description
Technical field
The invention belongs to gene engineering technology field, and in particular to one kind restructuring 4 albumen of archaerhodopsin and preparation method thereof and
Application in structure biology research.
Background technology
There are a kind of protein family to different wavelengths of light with different responses in the Nature, it is referred to as photosensitive protein.It
In the life process of biology a variety of important roles of performer, wherein most famous is exactly that crucial played in photosynthetic work makees
With.In view of its special function, not only biologist goes all out to study the relation of its 26S Proteasome Structure and Function, and material supply section scholar does not lose remaining yet
Its light-sensitive characteristic is directed to power, develops its application in terms of communication and light-sensitive material.In this protein family, bacterium regards
Rhodopsin (bacteriorhodopsin, BR) albumen is to be studied most deep, technology application and development most widely photosensitive egg
In vain, the existing research history of more than 30 years so far.Bacteria rhodopsin is a kind of thin positioned at the thermophilic salt bacillus H.salinarum of polarity
Seven-transmembrane protein on after birth, one retinene chromophore of covalent bond on its K216 positions.When BR light excites
Retinene chromophore forms a series of light intercycle states with different characteristic absorption peak there occurs change of configuration afterwards,
Thus there is photochromic characteristic.Since BR has the architectural characteristic of seven-transmembrane, with another kind of important film egg in organism
White g protein coupled receptor (GPCR) is similar, has been used as the research of GPCR structure biologies and molecular dynamics meter for a long time
The model of calculation.The BR of wild type forms stable tripolymer two-dimensional crystal lattice on thermophilic salt bacillus film, has better than most of
The high stability of large biological molecule, and to the light intercycle state of light sensitive and clear and definite characteristic, be significantly better than existing each
The photochromic material of kind chemical synthesis, makes it before optical information storage and optical information processing etc. have wide application
Scape.
Archaerhodopsin (Archaerhodopsin, AR) is the another kind of photosensitive protein similar with bacteria rhodopsin, it also has
There are proton-pump and light intercycle state.The nineties in last century, finds a kind of Halophiles in China's Zabuye Salt Lake In Tibet
Xz515, therefrom purifying obtain a kind of same memebrane protein with light cycle characteristics.By the amino acid sequence for analyzing this albumen
Row, the three kinds of archaerhodopsin albumen for finding and having confirmed that have the homology of height, therefore are named as archaerhodopsin 4 (AR4), and are China
It is exclusive.Although AR4 has proton-pump as BR, proton transfer order is not quite similar with BR, furthers investigate AR4's
Proton pump transporting mechanism is not only particularly significant to recognizing the transmission mechanism of its proton pump, at the same to explain BR proton transfers during
Still unapparent part, which also has, has very important significance.Importantly, the research of AR4 helps to develop the exclusive light letter in China
Breath storage and optical information processing material.To realize this target, the high efficient expression system of AR4 different functionalities Residue positions mutant
For just into the task of top priority.However, the method for the expression restructuring AR albumen of any type stabilization is not yet developed so far, more
It is far from being and obtains corresponding mutant.Have been reported document be related to method that AR4 genes express in L33 bacterial strains (Wang,
Y.et al.J.Fudan Nat.Sci.2003,42,576-583), but the method existing defects, its experiment knot can not be repeated out
Fruit.For its defect, the present invention modifies AR4 genes, while has also carried out part modification to the leading peptide gene of AR4,
Final successfully realize stablizes in L33, efficiently gives expression to restructuring AR4 albumen, and has constructed multiple mutant, is the later stage
A series of structure-function research work lay a good foundation.
Application of the retinene albumen in terms of Optical Information Storage and Processing, it is important to it is easily distinguishable photosensitive protein to be found
Two kinds of stable states.For example, bacterium visual purple feux rouges circulation M states absworption peak is located at 412nm, and ground state absorption peak is then 568nm, this
The distribution of sample is with regard to ideal.However, either the bacteria rhodopsin or archaerhodopsin of wild type, their light circulate very
Of short duration, wherein M states are shorter, only more than ten milliseconds, it is difficult to realize the storage and processing of optical information.But existing lot of documents report
To lead, some mutant of bacteria rhodopsin can greatly extend the decay of the decay, particularly M states of its different intermediate state,
And corresponding this part work of AR4 is since the hysteresis of expression system foundation is there is not yet report.Therefore, prepare and study AR4 mutation
The attenuation characteristic of body M states, the optical information storage exclusive to research and development China and optical information processing material equally have great reality
Meaning.
The content of the invention
The object of the present invention is to provide a kind of DNA recombinant expression wild type and saltant type seven-transmembrane protein to recombinate table
Up to 4 albumen of archaerhodopsin and its preparation method and application.
The present invention provides one kind to recombinate 4 albumen of archaerhodopsin, its its coding nucleotide sequence is as shown in SEQ ID NO.1；
Amino acid sequence is as shown in SEQ ID NO.2；Regulate and control the promoter sequence of 4 protein expression of archaerhodopsin as shown in SEQ ID NO.3.
Nucleotide sequence as shown in SEQ ID NO.1：
ATGTTGGAGTTATTGCCAACAGCAGTGATGGACCCGATAGCGCTACAGGCGGGATTCGACCTCCTCGGC
GATGGTCGTCCCGAGACACTCTGGTTGGGTATCGGCACGCTACTAATGATCATCGGGACCTTCTATTTCATCGCCCA
GGGGTGGGGCGTAACGGATAAGGAGGCCCGTGAGTACTACGCGATCACCATCCTCGTGCCGGGGATCGCGTCGGCCG
CGTATCTGGCGATGTTCTTCGGCATCGGTGTGACCGAAGTCGAACTCGCCAGCGGAGCGGTGCTCGACATCTACTAC
GCCCGGTACGCGGACTGGCTGTTCACGACGCCGCTGCTGCTGCTCGACCTCGCCCTGCTGGCGAAGGTTGACCGCGT
CAGCATCGGGACGCTCATCGGCGTCGACGCGCTGATGATCGTCACCGGTCTCATCGGCGCGCTCTCGAAGACGCCGC
TCGCGCGGTACACCTGGTGGCTGTTCAGCACGATCGCGTTCCTGTTCGTGCTGTACTACCTCCTGACGAGTCTGCGC
AGCGCCGCCGCGCAGCGCTCCGAAGAGGTCCAGAGTACCTTCAACACGCTGACCGCACTGGTCGCCGTCCTCTGGAC
GGCGTACCCGATCCTGTGGATTGTCGGGACCGAGGGCGCCGGCGTCGTTGGTCTGGGCGTCGAGACCCTGGCGTTCA
TGGTCCTCGACGTGACCGCGAAGGTCGGATTCGGGTTCGCCCTGCTCCGTAGCCGCGCGATCCTCGGCGAGACCGAG
GCCCCCGAGCCGTCGGCCGGCACTTGA
Amino acid sequence as shown in SEQ ID NO.2：
MLELLPTAVMDPIALQAGFDLLGDGRPETLWLGIGTLLMIIGTFYFIAQGWGVTDKEAREYYAITILVPGIASAAYL
AMFFGIGVTEVELASGAVLDIYYARYADWLFTTPLLLLDLALLAKVDRVSIGTLIGVDALMIVTGLIGALSKTPLAR
YTWWLFSTIAFLFVLYYLLTSLRSAAAQRSEEVQSTFNTLTALVAVLWTAYPILWIVGTEGAGVVG
LGVETLAFMVLDVTAKVGFGFALLRSRAILGETEAPEPSAGT
Promoter sequence as shown in SEQ ID NO.3：
CCAGTGAATTCGAGCTCGGTACCCGGGGATCCGAAGTGAAGATGGGGCTCCCGATGGGTGCAACCGTGA
AGTCCGTCACGGCTGCGTCACGACAGGAGCCGACCAGCGACACCCAGAAGGTGCGAACGGTTGAGTGCCGCAACGAT
CACGAGTTTTTCGTGCGCTTCGAGTGGTAACACGCGTGCACGCATCGACTTCACCGCGGGTGTTTCGACGCCAGCCG
GCCGTTGAACCAGCAGGCAGCGGGCATTTCACAGCCGCTGTGGCCCACACACTCGGTGGGGTGCGCTATTTTGGTAT
GGTTTGGAATCCGCGTGTCGGCTCCGTGTCTGACGGTTCATCGGTCTAAATTCCGTCACGAGCGTACCATACTGATT
GGGTCGTAGAGTTACACACATATCCTCGTTAGGTACTGTTGC
Restructuring 4 albumen of archaerhodopsin of the present invention is 4 albumen of archaerhodopsin of gene mutation, and the amino acid for modification of undergoing mutation is
Positioned at the aspartic acid of the 97th, belong to the important residue in archaerhodopsin april protein subchannel.The generation pair of this mutation
The light circulation of 4 albumen of archaerhodopsin, proton pump have a major impact, and mutant is to exist in the light intercycle service life of recombinant protein of the present invention
Have under the same terms compared with wild albumen and greatly extend.Compared with 4 albumen of archaerhodopsin of wild type extraction, present invention weight
4 albumen of group archaerhodopsin has the process of identical proton extraction and release, and with identical light circulation M states.Due to the present invention
4 albumen of archaerhodopsin of recombination expression can be widely applied to tie by that can obtain the high yield sample of milligram quantities level after purification
In structure biological study, for example, available for structure biology researchs such as X-ray crystallography, nuclear magnetic resonance spectroscopies.
Present invention also offers a kind of preparation method for recombinating 4 albumen of archaerhodopsin, it includes the following steps：
(1) gene of restructuring 4 albumen of archaerhodopsin is obtained；
With the plasmid pUC19-AR4 templates containing AR4 genes, with 5 '-GCCAACAGCAGTGATGGACCCGATAG-3 '
(SEQ ID NO：And 5 '-GCCAAGCTTCTAGATCAGTCGCTG-3 ' (SEQ ID NO 4)：5) it is primer, passes through PCR amplification
DNA fragmentation 1 is obtained, is then template with DNA fragmentation 1, with 5 '-ATGTTGGAGTTATTGCCAACAGCAGTG-3 ' (SEQ ID
NO：And 5 '-GCCAAGCTTCTAGATCAGTCGCTG-3 ' (SEQ ID NO 6)：5) it is primer, is recombinated by PCR amplification
4 protein nucleotide sequence of archaerhodopsin, as shown in SEQ ID NO.1.
(2) expression vector of structure restructuring 4 albumen of archaerhodopsin；
The nucleotide sequence as shown in SEQ ID NO.1 that step (1) is obtained, with the control as shown in SEQ ID NO.3
The promoter sequence of gene expression processed is spliced.The HindIII digestions at the ends of BamHI and 3 ' that spliced sequence passes through 5 ' ends
Site is connected with pUC19 plasmids, construction recombination plasmid pUC19-bAR；Through BamHI and HindIII double digestions, it is transferred to
On pXLNovR plasmids, structure forms expression vector pXLNovR-bAR plasmids；
(3) restructuring 4 albumen of archaerhodopsin is obtained；
PXLNovR-bAR plasmids are transferred in thermophilic salt bacillus (H.salinarum) L33 of host cell, conversion bacterial strain exists
In culture medium after culture, isolate and purify to obtain restructuring 4 albumen of archaerhodopsin.
The present invention utilizes technique for gene engineering, and 5 ' ends of wild AR4 genes are modified, and dashes forward to critical sites
Become, the gene after mutation is transferred in the bacterial strain for not producing retinene albumen, expresses archaerhodopsin mutant.Cultivated, separated by amplification,
Extraction, purifying obtain 4 albumen of mutation archaerhodopsin without bacterioruberin.
Preparation method of the present invention includes：
1. the gene overall length 789bp of wild type AR4, the leader peptide of preceding 57 alkali yl codings AR4 albumen.N-terminal 9 will be encoded
Gene (the 1-27 bit base sequences of peptide：5'-ATGCTATATGTAGATATGGGTATGGGT-3 ') replace with bop genes accordingly
Part (5'-ATGTTGGAGTTATTGCCAACAGCAGTG-3 '), replaced gene obtains mRNA by transcription, its 5 ' end
Loop-stem structure can be formed, promotes translation efficiency；
2. the AR4 genes after modification splice with bop gene promoters, bAR genes are named as, and pass through the BamHI at 5 ' ends
It is connected to form recombinant plasmid pUC19-bAR with pUC19 plasmids with the HindIII restriction enzyme sites at 3 ' ends, carries out DNA sequencing.Sequencing
The result shows that its sequence matches completely with objective gene sequence；
3. using pUC19-bAR plasmids as template, with KOD-Plus-Mutagenesis Kit (TOYOBO companies) kit
PUC19-bARD97N mutant plasmids are obtained, sequence is verified errorless by DNA sequencing；
4. it will be cloned on pUC19 plasmids and pass through the bAR genes of sequencing with the two limits of BamHI and HindIII
Property restriction enzyme site processed is transferred on pXLNovR plasmids, and the pXLNovR-bAR plasmids of genetic recombination are transferred to thermophilic salt bacillus
(H.salinarum) in L33；
5. conversion bacterial strain cultivates 12-16 days in the culture medium containing 0.5 μ g/ml ovobiocins, then picking color is most
Deep kermesinus bacterium colony 37 DEG C of shaken cultivations in the thermophilic salt bacillus peptone culture mediums of 10ml, after thalline color change, centrifugation
It is kermesinus to detect bacterial sediment color.Then amplification culture, separation, purifying step by step, finally obtains gene engineering expression not
4 albumen of restructuring archaerhodopsin containing bacterioruberin, i.e. bAR albumen.
Preparation method of the present invention includes：
1. using pUC19-bAR plasmids as template, with KOD-Plus-Mutagenesis Kit (TOYOBO companies) kit
PUC19-bARD97N mutant plasmids are obtained, sequence is verified errorless by DNA sequencing；
2. by be cloned on pUC19 plasmids and by sequencing mutation bAR genes with BamHI and HindIII this two
A restriction endonuclease sites are transferred on pXLNovR plasmids, and the pXLNovR-bAR plasmids of genetic recombination are transferred to thermophilic salt bar
In bacterium (H.salinarum) L33；
3. conversion bacterial strain cultivates 12-16 days in containing 0.5 μ g/ml ovobiocins, then the most deep bacterium colony of picking color
37 DEG C of shaken cultivations in the thermophilic salt bacillus peptone culture mediums of 10ml, after thalline color change, centrifugation detection bacterial sediment face
Color is kermesinus.Then amplification culture, separation, purifying step by step, finally obtains the D97N without bacterioruberin of gene engineering expression
It is mutated 4 albumen of archaerhodopsin.
Wherein, in 4 albumen of restructuring archaerhodopsin being prepared by the method for the present invention, the important amino acid of gene mutation modification is
Positioned at the aspartic acid of the 97th, the amino acid be in the sub- pump channel of archaerhodopsin april protein have to proton transfer it is important
The polar amino acid of effect.
By preparation method of the present invention can heterogenous expression it is wild or mutation 4 albumen of restructuring archaerhodopsin, the weight obtained
Group 4 albumen of archaerhodopsin has the function of identical with wild-type protein, and the albumen is free of bacterioruberin, eliminates the shadow of bacterioruberin
Ring, protein yield after purification can reach a milligram rank.Therefore 4 albumen of restructuring archaerhodopsin being prepared by the method for the present invention can use
In structure biology research.
Present invention also offers a kind of application for recombinating 4 albumen of archaerhodopsin in solid-state nuclear magnetic resonance detection.
In present invention application, in solid-state nuclear magnetic resonance detection, by being added in the culture medium of restructuring 4 albumen of archaerhodopsin
After the amino acid of isotope marks is cultivated, 4 albumen of restructuring archaerhodopsin of isotope marks is obtained after isolating and purifying.Preferably
Ground, in one embodiment, [U- is obtained after isolating and purifying13C9,15N]-Leu mark 4 albumen of restructuring archaerhodopsin.
The restructuring AR4 albumen of the isotope marks of milligram rank, the solid core of gained can be obtained by preparation method of the present invention
Magnetic resonance spectrogram shows that 4 albumen of restructuring archaerhodopsin is free of endophyte red pigment, and has good space folding, can further use
In structure biology research.
Present invention also offers application of restructuring 4 albumen of archaerhodopsin in Optical Information Storage and Processing.Made by the present invention
4 albumen of saltant type archaerhodopsin that Preparation Method obtains, its light circulation M lifetime of intermediate state occur greatly to extend, and opalescence is believed for this
The application of breath storage and processing etc. provides possibility.
The present invention passes through base using DNA recombinant expression wild type and the method for saltant type seven-transmembrane protein archaerhodopsin 4
Because of engineering technology, AR4 genes and promoter are suitably modified, and is transferred in thermophilic salt bacillus L33 and carries out protein expression.Through pure
Protein yield after change reaches milligram magnitude, and has the proton pump and light circulatory function identical with wild type AR4 albumen.To ancient purple
The amino acid that the light circulation of 4 albumen of matter and proton pump have a major impact carries out mutation modification, the mutation egg after to recombination expression
In vain after testing, its light circulates extension of the M state service lifes up to the order of magnitude.Mutain of the present invention, which recombinates 4 albumen of archaerhodopsin, makes AR4
Optical Information Storage and Processing function optimized, materials for binding biological research, available for preparing Optical Information Stovage and Processing
Membrane module.
Brief description of the drawings
Fig. 1 shows in embodiment 1, the bAR gene PCR amplified productions of recombination to construct, pass through 1% agarose gel electrophoresis
As a result.
Fig. 2 represents in embodiment 2 that restructuring 4 albumen of archaerhodopsin after purification is detected by 12% SDSPAGE, as a result with
Natural 4 albumen of archaerhodopsin compares.
Fig. 3 represents 4 albumen of restructuring wild type archaerhodopsin and 4 egg of wild type archaerhodopsin of wild type extraction in embodiment 3
White and wild type BR albumen proton-pumps comparison.Wherein, Fig. 3 (A) represents what is included by the bAR genes of recombination to construct
Key gene fragment；Fig. 3 (B) represents that bAR gene 5 's end carries the part identical with bop genes, can form loop-stem structure promotion
Protein expression；Fig. 3 (C) represents 4 albumen of archaerhodopsin of recombination expression and 4 albumen of natural archaerhodopsin have identical proton extraction with
The order of release.
It is bent that Fig. 4 represents to recombinate M states decay in 4 protein D 97N mutant light circular responses of wild type archaerhodopsin in embodiment 4
Line.
It is bent that Fig. 5 represents to recombinate M states decay in 4 protein Y 186F mutant light circular responses of wild type archaerhodopsin in embodiment 5
Line.
Fig. 6 represents [U- in embodiment 613C9,15N]-Leu mark restructuring 4 albumen of archaerhodopsin solid-state nuclear magnetic resonance bidimensional
Single quantum state related experiment result (Fig. 6 (A)) and the double quantum state-single quantum state phases of BR bidimensionals with identical residue isotope marks
The superposition for closing experimental result (Fig. 6 (B)) is compared.
Embodiment
With reference to specific examples below and attached drawing, the present invention is described in further detail, protection content of the invention
It is not limited to following embodiments.Without departing from the spirit and scope of the invention, those skilled in the art it is conceivable that change
Change and advantage is all included in the present invention, and using appended claims as protection domain.Do not noted in the following example
The experimental method of bright actual conditions, usually according to normal condition such as Sambrook et al., molecular cloning：Lab guide (New
York：Cold Spring Harbor Laboratory Press, 1989) condition described in, or built according to manufacturer
The condition of view.Unless otherwise stated, otherwise percentage and number are calculated by weight.Implement the present invention process, condition, reagent,
Experimental method etc., in addition to the following content specially referred to, is among the general principles and common general knowledge in the art, the present invention does not have
Especially limitation content.
Embodiment 1：
Using wild type AR4 genes as template, 5 ' end 1-27 bit bases of AR4 genes are replaced, specific method is as follows：
Using the plasmid pUC19-AR4 containing wild type AR4 genes as template, with 5'-GCCAACAGCAGTGATGGACCCGATAG-3'
(SEQ ID NO：And 5'-GCCAAGCTTCTAGATCAGTCGCTG-3 ' (SEQ ID NO 4)：5) it is primer, passes through Pfu DNA
Polymerase PCR amplification obtains DNA fragmentation 1, is then template with DNA fragmentation 1, with 5'-
ATGTTGGAGTTATTGCCAACAGCAGTG-3’(SEQ ID NO：And 5'-GCCAAGCTTCTAGATCAGTCGCTG-3 ' 6)
(SEQ ID NO：5) it is primer, restructuring 4 nucleotide sequence of archaerhodopsin, such as SEQ is obtained by Pfu archaeal dna polymerase PCR amplifications
Shown in ID NO.1.
Then, 4 gene of archaerhodopsin will be recombinated with regulating and controlling the promoter sequence of its expression to be spliced, step is as follows：With containing
The pUC19-bop plasmids of purposeful promoter sequence are template, with 5'-CGGGATCCGACGTGAAGATGGGG-3 ' (SEQ ID
NO：And 5'-ACTCCAACATGCAACAGTACCTAAC-3 ' (SEQ ID NO 7)：8) it is primer, passes through Pfu archaeal dna polymerases
PCR amplification obtains bop promoter fragments.To recombinate 4 gene of archaerhodopsin as template, with 5'-
GTTAGGTACTGTTGCATGTTGGAGT-3’(SEQ I D NO：And 5'-GCCAAGCTTCTAGATCAGTCGCTG-3 ' 9)
(SEQ ID NO：5) it is primer, DNA fragmentation 2 is obtained by Pfu archaeal dna polymerase PCR amplifications.The bop that above-mentioned amplification is obtained
Promoter fragment and DNA fragmentation 2 mix, with 5'-CGGGATCCGACGTGAAGATGGGG-3' and 5'-
GCCAAGCTTCTAGATCAGTCGCTG-3’(SEQ I D NO：5) it is primer, the second wheel is carried out by Pfu archaeal dna polymerases
PCR, amplification obtain the gene order that a both ends are respectively provided with the 1.2kb of BamHI and HindIII restriction sites, are named as
bAR.PCR product passes through 1% agarose gel electrophoresis, a visible obvious band at 1.2kb, with the big rostellum of expected product
Close, as shown in Figure 1.BAR gene of the gel extraction after PCR amplification, with BamHI and HindIII double digestions bAR and pUC19 matter
After grain, conventionally connected with T4 ligases, and be transformed into Top10 competent cells.Selected at random on tablet several
Clone, extracts plasmid, the accuracy for determining target gene bAR is sequenced by company, sequencing result is consistent with design, will newly build
Plasmid be named as pUC19-bAR.
Embodiment 2
Gene mutation described in the present invention is by KOD-Plus-Mutagenesis Kit (TOYOBO companies) reagent
What the method that box provides was realized, comprise the following steps that：Using pUC19-bAR plasmids as template, two inverse PCR primers are designed, this
Two primer available linearization template plasmids after PCR amplification, wherein 5 ' ends of a primer are with the password for being intended to Mutated residues
Son.With this two primers, PCR amplification is carried out to template plasmid with KOD archaeal dna polymerases, obtains the fragment of 3800bp, this fragment
Mutational site is carried.PCR conditions are 94 DEG C, 2min, 98 DEG C, 10sec, 68 DEG C, 4min, 15 circulations.Add in PCR product
Enter DpnI restriction enzymes, when 37 DEG C of processing 1 are small, digested plasmid template.Postdigestive PCR product is carried out by phosphokinase
Phosphorylation, then realizes recirculation with ligase.Product conversion Escherichia coli Top10 competent cells after cyclisation,
Several clones are selected on tablet at random, extract plasmid, the accuracy for determining mutator is sequenced by company.Its in example 2
His details, refers to the specification of KOD-Plus-Mutagenesis Kit (TOYOBO companies) kit offer.
Embodiment 3：
Using the method described in embodiment 1,5 ' ends of wild AR4 genes are modified, enable the gene after modification
Enough expression restructuring 4 albumen of archaerhodopsin in the bacterial strain for not producing retinene albumen.The gene overall length 789bp of wild type AR4, first 57
The leader peptide of alkali yl coding AR4 albumen.The gene for encoding 9 peptides of N-terminal is replaced with into corresponding part in bop genes, after replacement
Gene obtain mRNA by transcription, its 5 ' end can form loop-stem structure, promote translation efficiency (Fig. 3 B).AR4 bases after modification
Because splicing with bop gene promoters, bAR genes (Fig. 3 A), and the HindIII enzymes at the ends of BamHI and 3 ' for passing through 5 ' ends are named as
Enzyme site is connected to form recombinant plasmid pUC19-bAR with pUC19 plasmids, carries out DNA sequencing.Sequencing result shows its sequence and mesh
Gene order match completely.It will be cloned on pUC19 plasmids and pass through the bAR genes of sequencing with BamHI and HindIII
The two restriction endonuclease sites are transferred on pXLNovR expression plasmids.By protoplast transformation method, by genetic recombination
PXLNovR-bAR plasmids be transferred in thermophilic salt bacillus (H.salinarum) L33.Bacterial strain is converted newborn containing 0.5 μ g/ml
Cultivated in the culture medium of mycin 12-16 days, then the most deep kermesinus bacterium colony of picking color is in the thermophilic salt bacillus peptone trainings of 10ml
37 DEG C of shaken cultivations in base are supported, after thalline color change, centrifugation detection bacterial sediment color is kermesinus.Then amplify step by step
Culture, the absorbance at 660nm harvest bacterium after being more than 1.2.4000 × g centrifuges 20 minutes harvest thalline, after centrifugation
Product is resuspended with the NaCl solution of 4M, and the NaCl solution of 100mM is dialyzed overnight.Product after dialysis passes through 40000
× g is centrifuged 40 minutes, abandons supernatant, precipitation is resuspended with deionized water.Protein crude extract administration after resuspension by sucrose gradient centrifugation come
It is further purified, finally obtains 4 albumen of restructuring archaerhodopsin without bacterioruberin of gene engineering expression, be named as bAR albumen.It is logical
12% SDSPAGE detections are crossed, display bAR molecular weight of albumen is about 26kD, (Fig. 2) identical with natural AR4 albumen.Pass through reality
The self-built chemical kinetic systems instrument in room is tested to be detected the function of bAR albumen, as a result as shown in Figure 3 C, the archaerhodopsin of recombination expression
4 albumen and order of 4 albumen of archaerhodopsin with identical proton extraction and release that wild type is extracted, and carried with the proton of BR
Take-release order it is opposite.
Embodiment 4：
4 egg of archaerhodopsin that the 97th aspartic acid (Asp) be mutated into asparagine (Asn) is obtained using site-directed mutagenesis technique
White gene.Using pUC19-bAR plasmids as template, obtained with KOD-Plus-Mutagenesis Kit (TOYOBO companies) kit
PUC19-bARD97N mutant plasmids are obtained, sequence is verified errorless by DNA sequencing.It will be cloned on pUC19 plasmids and pass through sequence
With BamHI and HindIII, the two restriction endonuclease sites are transferred on pXLNovR plasmids the mutation bAR genes of measure, will
The pXLNovR-bAR plasmids of genetic recombination are transferred in thermophilic salt bacillus (H.salinarum) L33.Conversion bacterial strain is containing 0.5 μ
Cultivated in the culture medium of g/ml ovobiocins 12-16 days, then the most deep bacterium colony of picking color is in the thermophilic salt bacillus peptones of 10ml
37 DEG C of shaken cultivations in culture medium, after thalline color change, centrifugation detection bacterial sediment color is kermesinus.Then put step by step
Big culture, and Protein Separation and purifying are carried out by the method described in embodiment 3, finally obtain gene engineering expression is free of bacterium
D97N mutation 4 albumen of archaerhodopsin of red pigment.The function of mutain, is examined by the self-built chemical kinetic systems instrument in laboratory
Survey, the results are shown in Figure 4, and the decay of its M state occurs greatly to extend.
Embodiment 5：
4 egg of archaerhodopsin that the 186th tyrosine (Tyr) be mutated into phenylalanine (Phe) is obtained using site-directed mutagenesis technique
White gene.Using pUC19-bAR plasmids as template, obtained with KOD-PIus-Mutagenesis Kit (TOYOBO companies) kit
PUC19-bARY186F mutant plasmids are obtained, sequence is verified errorless by DNA sequencing.It will be cloned on pUC19 plasmids and pass through sequence
With BamHI and HindIII, the two restriction endonuclease sites are transferred on pXLNovR plasmids the mutation bAR genes of row measure,
The pXLNovR-bAR plasmids of genetic recombination are transferred in thermophilic salt bacillus (H.salinarum) L33.Conversion bacterial strain is containing 0.5
Cultivated in the culture medium of μ g/ml ovobiocins 12-16 days, then the coloured bacterium colony of picking band is in the thermophilic salt bacillus peptones of 10ml
37 DEG C of shaken cultivations in culture medium, after thalline color change, centrifugation detection bacterial sediment color is pale red.Then put step by step
Big culture, and Protein Separation and purifying are carried out by the method described in embodiment 3, finally obtain gene engineering expression is free of bacterium
Y186F mutation 4 albumen of archaerhodopsin of red pigment.The function of mutain, is examined by the self-built chemical kinetic systems instrument in laboratory
Survey, the results are shown in Figure 5, its M state service life is more slightly longer than wild AR4 albumen.
Embodiment 6：
In AR4 incubations in embodiment 1,2 or 3, respectively by the bright ammonia of nonisotopic labels in synthetic media
Acid is substituted for13C,15The leucine of N isotope marks, culture restructuring 4 albumen of archaerhodopsin and BR albumen.By amplifying training step by step
Support, isolate and purify to obtain [U-13C9,15N]-Leu-bAR leucines mark 4 albumen of restructuring archaerhodopsin and BR albumen, and carry out
Two kinds of different bidimensional solid-state nuclear magnetic resonance experiments.
Thermophilic salt bacillus synthetic media component, it is as follows：
Amino acid moiety：
Inorganic salts part：
Trace metal ion
Other materials：
Adenylic acid 100mg/L
Uridylic acid 100mg/L
Glycerol 1g/L
Solid-state nuclear magnetic resonance experimental result is as shown in fig. 6, due to comprising more than 30 leucines in AR4, and widely
It is distributed in TM1-TM7, there is similar chemical environment.The different resonance frequencies that bidimensional single quantum state related experiment result is shown
The relatively narrow peak width of rate illustrates that self-assembly of the restructuring archaerhodopsin 4 in L33 in raw phosphatide is correct, and protein folding is normal and with good
Space conformation, as shown in Fig. 6 (A).Double quantum state-the single quantum states of bidimensional for recombinating 4 albumen of archaerhodopsin and BR albumen are related real
Test and show shown in result such as Fig. 6 (B), what wherein black contour represented is the experimental result for recombinating 4 albumen of archaerhodopsin, Dark grey
What contour represented is the experimental result of BR albumen, and what * was indicated is spinning side band.Use double quantum state filtering technologies can be with
Only obtain isotope marks residue structural information, can clearly be identified from spectrogram magnetization vector from CO → C α → C β →
The transfer of C γ → C δ, all spectrum peak positions that restructuring 4 albumen of archaerhodopsin and BR albumen chart addings obtain are completely overlapped, explanation
The phosphatide for recombinating 4 albumen of archaerhodopsin and BR albumen forms consistent, consistent to the modulating action of albumen, is free of in L33 in raw phosphatide
There is bacterioruberin.Above solid-state nuclear magnetic resonance experimental result can absolutely prove, using the restructuring archaerhodopsin 4 of the method for the present invention preparation
Albumen can be used for the structure-biological science study method such as nuclear magnetic resonance or X-ray diffraction parsing Membrane protein conformation and study its structure-
Functional relationship.
Claims (4)
1. one kind restructuring 4 albumen of archaerhodopsin, it is characterised in that the coding nucleotide sequence such as SEQ of restructuring 4 albumen of archaerhodopsin
Shown in ID NO.1, its amino acid sequence is as shown in SEQ ID NO.2.
2. a kind of preparation method of 4 albumen of archaerhodopsin of restructuring as claimed in claim 1, it is characterised in that include the following steps：
(1) gene of restructuring 4 albumen of archaerhodopsin is obtained；
Using the plasmid pUC19-AR4 containing AR4 genes as template, with 5'-GCCAACAGCAGTGATGGACCCGATAG-3'(SEQ
ID NO：And 5'-GCCAAGCTTCTAGATCAGTCGCTG-3 ' (SEQ ID NO 4)：5) it is primer, is obtained by PCR amplification
DNA fragmentation 1, is then template with DNA fragmentation 1, with 5'-ATGTTGGAGTTATTGCCAACAGCAGTG-3 ' (SEQ ID NO：
And 5'-GCCAAGCTTCTAGATCAGTCGCTG-3 ' (SEQ ID NO 6)：5) it is primer, obtains recombinating ancient purple by PCR amplification
4 protein nucleotide sequence of matter, as shown in SEQ ID NO.1；
(2) expression vector of structure restructuring 4 albumen of archaerhodopsin；
The nucleotide sequence as shown in SEQ ID NO.1 that step (1) is obtained, with the control base as shown in SEQ ID NO.3
Because the promoter sequence of expression is spliced；The HindIII restriction enzyme sites at the ends of BamHI and 3 ' that spliced sequence passes through 5 ' ends
It is connected with pUC19 plasmids, construction recombination plasmid pUC19-bAR；Through BamHI and HindIII double digestions, pXLNovR matter is transferred to
On grain, structure forms expression vector pXLNovR-bAR plasmids；
(3) restructuring 4 albumen of archaerhodopsin is obtained；
PXLNovR-bAR plasmids are transferred in the thermophilic salt bacillus L33 of host cell and carry out protein expression, conversion bacterial strain is in culture medium
It is middle after culture, isolate and purify to obtain restructuring 4 albumen of archaerhodopsin.
3. preparation method as claimed in claim 2, it is characterised in that 4 albumen of restructuring archaerhodopsin that the preparation method obtains
Without bacterioruberin.
4. a kind of application of restructuring 4 albumen of archaerhodopsin as claimed in claim 1 in solid-state nuclear magnetic resonance detection, its feature exist
In, by restructuring 4 albumen of archaerhodopsin culture medium in add isotope marks amino acid cultivated, isolate and purify to obtain
4 albumen of restructuring archaerhodopsin of isotope marks.
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