CN103215235B - 3-imidazolyl tyrosine translation system and use thereof - Google Patents
3-imidazolyl tyrosine translation system and use thereof Download PDFInfo
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- CN103215235B CN103215235B CN201210015550.1A CN201210015550A CN103215235B CN 103215235 B CN103215235 B CN 103215235B CN 201210015550 A CN201210015550 A CN 201210015550A CN 103215235 B CN103215235 B CN 103215235B
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Abstract
The invention relates to an aminoacyl-tRNA synthetase mutant. The aminoacyl-tRNA synthetase mutant has an amino acid sequence composed of amino acids shown in the formula of SEQ ID NO.2 and their conservative mutants. The invention provides a 3-imidazolyl tyrosine translation system for specific and fixed-point insertion of 3-imidazolyl tyrosine (imiTyr) into a target protein by utilization of an orthogonal tRNA, an orthogonal aminoacyl-tRNA synthetase and their complex, and also provides a method for specific and fixed-point insertion of the 3-imidazolyl tyrosine into the target protein by the 3-imidazolyl tyrosine translation system. The 3-imidazolyl tyrosine translation system comprises 1), 3-imidazolyl tyrosine, 2), the orthogonal aminoacyl-tRNA synthetase, 3), the orthogonal tRNA, wherein the orthogonal aminoacyl-tRNA synthetase is obtained by preferential aminoacylation of the orthogonal tRNA by the 3-imidazolyl tyrosine, and 4), a nucleic acid for encoding a target protein, wherein the nucleic acid contains at least one selection codons specifically recognized by the orthogonal tRNA. The invention also relates to a method for specific and fixed-point insertion of 3-imidazolyl tyrosine in Myoglobin (Mb) so that the Myoglobin has oxidase activity, and a use of the method.
Description
Technical field
The invention belongs to biochemical field.Particularly, the invention provides aminoacyl-tRNA synthetase mutant, the group that its aminoacid sequence containing selects amino acid shown in free SEQ ID NO:2 and their examples of conservative variations to form.The invention still further relates to a kind of 3-imidazolyl tyrosine ((S)-2-amino-3-(4-hydroxyl-3-(1H-imidazoles-1-yl) phenyl) propionic acid, is abbreviated as imiTyr) translation system.More specifically; the present invention relates to utilize orthogonal tRNA, orthogonal aminoacyl-tRNA synthetase and their pairing by the 3-imidazolyl tyrosine 3-imidazolyl tyrosine translation system of special insertion target protein of fixing a point, and utilize the fix a point method of special insertion 3-imidazolyl tyrosine of described translation system in target protein.The invention still further relates to the mutein that contains 3-imidazolyl tyrosine producing by this translation system and this method, for example, the myohaemoglobin mutant that contains 3-imidazolyl tyrosine, and the application of the mutein that contains 3-imidazolyl tyrosine.
Background technology
Protein is the essential substance basis of all life entities and vital movement thereof.In whole albumen, approximately having 1/3rd albumen is all some metallic albumen, is called metalloprotein (Metalloprotein) or metalloenzyme (Metalloenzyme).Reduced hematin (Hemoproteins) be exactly wherein a large class contain protoporphyrin IX (protoheme, heme) as the metalloprotein of prothetic group, in life system, carrying out important biological function, as oxygen carrier function (Hb H b and myohaemoglobin Mb), electron transfer function (cytochrome b5, cyt b5 and cytochrome c, cyt c etc.), biocatalytic Activity (Cytochrome P450, cyt P450 cytochrome c peroxidase, CcP etc.), and bio-sensing function (CO sensor CooA and NO sensor sGC etc.).Although reduced hematin has widely different biological function, they have almost similar protoheme prothetic group, and the protein molecular building form of similar protein and peptide chain parcel protoheme.How identical heme prothetic group is utilized by different protein moleculars, carries out different biological functions, is the emphasis that chemicobiology and protein chemistry research field are paid close attention to always.The researcher of lot of domestic and foreign has carried out research for a long time and widely, attempt to answer this problem, and by the answer of this problem being familiar with to the exquisite relation containing between the structure-character-reaction-function (structure-property-reactivity-function, SPRF) of albumen.
Protoheme-copper oxydase (HCOs) is the dual combination center protein that existing protoheme has again copper, is that a large class is extensively present in the terminal oxidase on eucaryon plastosome and bacterial respiratory chain.This proteinoid can be accepted four electronics from substrate, and is delivered on an oxygen molecule, oxygen is finally converted into two water moleculess, and in this process, can produce active oxygen (ROS).Wherein protoheme in conjunction with center can with an axial Histidine coordination, and copper in conjunction with center (CuB) can with three contiguous Histidine coordinations, cupric ion in the heart and iron ion are apart from approximately in two
scientists is mainly passed through structure biology at present, zymetology, spectrum, structure and the functions of method to HCO such as x-ray and plan ecological model are studied, and obtain huge breakthrough, but still existed the associated problem of some important structure and mechanism aspects to need further to inquire into.For example, CuB in conjunction with and activate aspect oxygen to play what effect actually? at protoheme-copper in conjunction with center, the reduction of oxygen concentration is transmission or the picked-up of associated proton how? the posttranslational modification that high-resolution X-ray structure has disclosed HCO makes tyrosine C6 and adjacent Histidine N ε 2 form covalent cross-linking, and in bacterium and Mammals, the covalent cross-linking structure of this Tyr-His is all guarded, confirm that it necessarily has critical function, but also Nobody Knows for the current exact function about this structure.
In order further to understand CuB and the function of Tyr-His covalent cross-linking in HCO, there is people to simulate HCO albumen by introduce copper center in yeast cell pigment c oxydase and Physter macrocephalus myohaemoglobin.Wherein myohaemoglobin (Myoglobin, Mb) formed by 153 amino acid and a protoheme prothetic group, have that molecular weight is little, stability is high, easily obtain the features such as high purity protein by genetically engineered and protein engineering, extremely investigator's parent looks at always for a long time, is extensively used for particularly reduced hematin structure and function relation of Study on Protein as the model of protein molecule.The biological function of Mb is to store and transport oxygen molecule, is typical oxygen carrier albumen.The prothetic group heme that contains a high-spin pentacoordinate in its molecule, the 5th axial ligand is His93, and a hole of the 6th axial existence, can be reversibly in conjunction with O
2molecule.Meanwhile, around two conservative amino-acid residue His64 and Val68 can stablize O in hole
2the existence of molecule.At present, people progressively recognize that the difference of reduced hematin biological function depends primarily on the difference on protoheme active sites, comprise the coordination situation of protoheme center iron, the microenvironment of protoheme binding cavity, and the mode of action between protoheme and protein peptide chain etc.For the difference in the intermolecular structure of these naturally occurring reduced hematins and function, it is not insurmountable barrier.Growing along with genetically engineered and protein engineering, now, people can carry out the design & formulation of protein molecular reasoningly, do suitably to modify and adjust by the active sites to reduced hematin, thereby realize the mutual conversion between different reduced hematin biological functions, as the mutual conversion between mutual conversion, electron transfer protein and biological catalyst between mutual conversion, oxygen carrier and biological catalyst between oxygen carrier and electron transfer protein, and mutual conversion between dissimilar reduced hematin etc.
In reduced hematin molecule, introducing other metal ion binding site, is a bright spot in reduced hematin structure and function study on the transformation field.By Mb and the oxidasic active sites of protoheme-copper are done to careful comparison, amino acid Leu29 and Phe43 in hole, Mb active centre are mutated into His, in addition the far-end part His64 originally existing in hole, in obtained mutant Mb L29H/F43H molecule, just there are 3 His in its active centre, they can be in conjunction with a Cu
2+ion (being called CuBMb).Existing investigator confirms through experiment, this mutant due to lack proton and an electronics pipeline and at O
2in conjunction with making heme degraded in reduction process, thereby also reflect from the side the importance of the covalent cross-linking of Tyr-His in protoheme-copper oxydase HCO.3-imidazolyl tyrosine has the characteristic group of Tyr-His covalent cross-linking, and imidazolyl and 3 carbon of tyrosine is covalently bound.For the further structure and function of these protein of research, this area also need this non-natural 3-imidazolyl tyrosine to be fixed a point new departure of special insertion protein.Now develop in protokaryon and eukaryote and will in various alpha-non-natural amino acid bodies, fix a point to insert the universal method of protein locus specificity.These methods depend on orthogonal protein translation component, and described component identifies suitable selection codon (selector codon) thereby polypeptide translate duration inserts required alpha-non-natural amino acid to limit position in vivo.These methods are utilized the orthogonal tRNA (O-tRNA) of identification selection codon, and the corresponding orthogonal aminoacyl-tRNA synthetase of specificity (O-RS) loads this O-tRNA with alpha-non-natural amino acid.These components not with host organisms in any endogenous tRNA, aminoacyl-tRNA synthetase (RS), amino acid or codon cross reaction (, it must be orthogonal).Utilize the alpha-non-natural amino acid of a large amount of configurations of this orthogonal tRNA-RS pairing possibility genetic coding.
This area generally knows that utilization is suitable for the orthogonal translation system of preparation containing the protein of one or more alpha-non-natural amino acids, for example, produce the universal method of orthogonal translation system.For example, referring to international publication number WO 2002/086075, its denomination of invention is " METHODS AND COMPOSITION FOR THE PRODUCTION OF ORTHOGONAL tRNA-AMINOACYL-tRNA SYNTHETASE PAIRS "; WO 2002/085923, its denomination of invention is " IN VIVO INCORPORATION OF UNNATURAL AMINO ACIDS "; WO 2004/094593, its denomination of invention is " EXPANDING THE EUKARYOTIC GENETIC CODE ".The orthogonal translation system of special insertion alpha-non-natural amino acid of fixing a point and other discussion of their generation and using method also can be referring to Wang and Schultz, Chem.Commun. (Camb) 1:1-11 (2002); Wang and Schultz, Angewandte Chemie Int.Ed.44 (1): 34-66 (2005); Xie and Schultz, Methods 36 (3): 227-238 (2005); Xie and Schultz, Curr.Opinion in Chemical Biology 9 (6): 548-554 (2005); Wang etc., Annu.Rev.Biophys.Biomol.Struct.35:225-249 (2006).
Summary of the invention
1, technical problem
The invention provides aminoacyl-tRNA synthetase mutant, the group that its aminoacid sequence containing selects amino acid shown in free SEQ ID NO:2 and their examples of conservative variations to form.The present invention relates to utilize orthogonal tRNA, orthogonal aminoacyl-tRNA synthetase and their pairing by the 3-imidazolyl tyrosine 3-imidazolyl tyrosine translation system of special insertion target protein of fixing a point, and utilize the fix a point method of special insertion 3-imidazolyl tyrosine of described translation system in target protein.The invention still further relates to the mutein and the application thereof that contain 3-imidazolyl tyrosine that produce by this translation system and this method.
Therefore; the object of the present invention is to provide and utilize orthogonal tRNA, orthogonal aminoacyl-tRNA synthetase and their pairing by the 3-imidazolyl tyrosine 3-imidazolyl tyrosine translation system of special insertion protein of fixing a point, and the method that provides this translation system to fix a point special insertion 3-imidazolyl tyrosine in target protein.
The present invention also provides the mutein that contains at least one 3-imidazolyl tyrosine that utilizes 3-imidazolyl tyrosine translation system of the present invention to produce.In preferred aspects of the invention, inventor's profit is fixed a point 3-imidazolyl tyrosine in special insertion myohaemoglobin (Myoglobin) in this way, and this albumen, by introducing cupric ion in conjunction with center, can produce oxidase activity.But, it should be appreciated by those skilled in the art that method of the present invention also can, for the special insertion 3-imidazolyl tyrosine of fixing a point in the multiple protein outside myohaemoglobin, be not limited to this albumen.
2, technical scheme
The inventor, through screening, obtains a kind of orthogonal aminoacyl-tRNA synthetase, the group that its aminoacid sequence containing selects amino acid shown in free SEQ ID NO:2 and their examples of conservative variations to form.And the inventor utilizes described orthogonal aminoacyl-tRNA synthetase, research and develop 3-imidazolyl tyrosine translation system.
Specifically, the invention provides in vivo (for example, in host cell) to selecting codon (selector codon) to react and by the fix a point 3-imidazolyl tyrosine translation system of the special insertion polypeptide chain in extending of alpha-non-natural amino acid 3-imidazolyl tyrosine as amber terminator codon (TAG).Described 3-imidazolyl tyrosine translation system comprise not with host cell translating mechanism interactional orthogonal-tRNA (O-tRNA) and orthogonal aminoacyl-tRNA synthetase (O-RS) pairing., host cell endogenous aminoacyl-tRNA synthetase can not use amino acid (natural or non-natural) to load O-tRNA.Similarly, O-RS provided by the invention not with in conspicuous level or some situation not with can detection level load endogenous tRNA with amino acid (natural or non-natural).Utilize described translation system to produce to contain a large amount of protein of special insertion 3-imidazolyl tyrosine of fixing a point in translation process.
In certain aspects, the invention provides 3-imidazolyl tyrosine translation system.Described translation system comprises: (a) alpha-non-natural amino acid; be 3-imidazolyl tyrosine; (b) orthogonal aminoacyl-tRNA synthetic enzyme (O-RS); (c) orthogonal tRNA (O-tRNA); it comprises the polynucleotide sequence shown in SEQ ID NO:1; wherein said orthogonal aminoacyl-tRNA is described alpha-non-natural amino acid (being 3-imidazolyl tyrosine) for synthetic enzyme, preferentially O-tRNA described in aminoacylation.
Preferably, 3-imidazolyl tyrosine translation system of the present invention also comprises the nucleic acid of the target protein of encoding, and wherein said nucleic acid contains at least one the selection codon by orthogonal tRNA (O-tRNA) specific recognition, is preferably amber codon.More preferably, 3-imidazolyl tyrosine translation system of the present invention also comprises the nucleotide sequence of coded orthogonal aminoacyl-tRNA synthetase.
In described system, orthogonal aminoacyl-tRNA synthetase (O-RS) used is the aminoacyl tRNA synthetase mutant that the inventor finds, the group that its aminoacid sequence containing selects amino acid shown in free SEQ ID NO:2 and their examples of conservative variations to form.
In preferred aspects of the invention, the invention provides a kind of 3-imidazolyl tyrosine translation system, described system comprises:
(i) 3-imidazolyl tyrosine;
(ii) orthogonal aminoacyl-tRNA synthetase;
(iii) orthogonal tRNA, it comprises the polynucleotide sequence shown in SEQ ID NO:1; Orthogonal tRNA described in the preferential aminoacylation of described 3-imidazolyl tyrosine for wherein said orthogonal aminoacyl-tRNA synthetase; With
(iv) nucleic acid of coding target protein, at least one selection codon that wherein said nucleic acid contains described orthogonal tRNA specific recognition.
Preferably, described 3-imidazolyl tyrosine translation system also comprises the nucleotide sequence of coded orthogonal aminoacyl-tRNA synthetase.
Various components in this translation system can be derived from various source of species, and for example, the each component in this translation system is derived from Methanococcus jannaschii (Methanococcus jannaschii).For example, the anticodon that orthogonal tRNA (O-tRNA) originates for ancient bacterium sports the tyrosine tRNA with amber codon complementation.In some embodiments, O-tRNA is amber inhibition type tRNA.In some embodiments, O-tRNA comprises the polynucleotide sequence shown in SEQ ID NO:1, and preferably, the sequence of O-tRNA is as shown in SEQ ID NO:1.In one embodiment, can comprise the aminoacid sequence shown in SEQ ID NO:2 and the conservative variant of this sequence for the orthogonal aminoacyl-tRNA synthetase (O-RS) of this system.
In certain aspects, 3-imidazolyl tyrosine translation system of the present invention also comprises the nucleic acid of the target protein of encoding, and wherein said nucleic acid has at least one the selection codon by orthogonal tRNA (O-tRNA) specific recognition.In aspect preferably, described orthogonal tRNA is amber inhibition type tRNA, and described selection codon is amber codon.
In certain aspects, the invention provides the host cell of the nucleotide sequence that comprises orthogonal tRNA sequence and coded orthogonal aminoacyl-tRNA synthetase.Host cell used is not done concrete restriction, as long as O-RS and O-tRNA retain their orthogonality in their host cell environment.For example, described host cell can be eubacterium cell, as intestinal bacteria.As described embodiments, can be by the recombinant vectors cotransformation of the recombinant vectors that comprises orthogonal tRNA sequence and the nucleotide sequence that comprises coded orthogonal 3-imidazolyl tyrosine aminoacyl-tRNA synthetase in host cell, and obtain the host cell of the nucleotide sequence that comprises orthogonal tRNA sequence and coded orthogonal 3-imidazolyl tyrosine aminoacyl-tRNA synthetase.
The present invention also provides the method for the mutein that is created in the special insertion of at least one selected location fixed point 3-imidazolyl tyrosine.Described method is utilized above-mentioned 3-imidazolyl tyrosine translation system.Described method starts from the step that the 3-imidazolyl tyrosine translation system that contains following component is provided conventionally: (i) alpha-non-natural amino acid, i.e. 3-imidazolyl tyrosine; (ii) orthogonal aminoacyl-tRNA synthetase (O-RS); (iii) orthogonal tRNA (O-tRNA), it comprises the polynucleotide sequence shown in SEQ ID NO:1, O-tRNA described in the preferential aminoacylation of described alpha-non-natural amino acid for wherein said O-RS (being 3-imidazolyl tyrosine); (iv) nucleic acid of coding target protein, at least one selection codon (being optionally amber codon) that wherein said nucleic acid contains O-tRNA specific recognition; Then the nucleic acid of the described target protein of coding is transformed in the host cell of the nucleotide sequence that comprises orthogonal tRNA sequence and coded orthogonal aminoacyl-tRNA synthetase; in the translation process of described protein; the O-tRNA of 3-imidazolyl tyrosine aminoacylation reacts to described selection codon and by the selected location of the described target protein of special insertion of fixing a point of the 3-imidazolyl tyrosine in substratum, thereby is created in the mutein that 3-imidazolyl tyrosine is contained in selected location.The host cell that wherein comprises the nucleotide sequence of orthogonal tRNA sequence and coded orthogonal 3-imidazolyl tyrosine aminoacyl-tRNA synthetase can be by obtaining the recombinant vectors that comprises orthogonal tRNA sequence and the recombinant vectors cotransformation of the nucleotide sequence that comprises coded orthogonal 3-imidazolyl tyrosine aminoacyl-tRNA synthetase in selected host cell.It should be appreciated by those skilled in the art that this can be by conventional molecule clone technology and triage techniques realization.
In some embodiments of described method; providing the step of translation system to comprise by site-directed mutagenesis undergos mutation the amino acid binding pocket of wild-type aminoacyl-tRNA synthetase; select the aminoacyl-tRNA synthetase mutant (, the present invention's orthogonal aminoacyl-tRNA synthetase used) with O-tRNA described in the preferential aminoacylation of described alpha-non-natural amino acid (being 3-imidazolyl tyrosine).Described selection step is carried out just selection and the negative selection (referring to following embodiment 2) of described O-RS from the aminoacyl-tRNA synthetase library of molecules obtaining after comprising site-directed mutagenesis.In some embodiments, provide the step of translation system also to comprise the sequence that O-tRNA is provided, O-tRNA is that the anticodon in ancient bacterium source sports and the tyrosine tRNA of amber codon complementation, for example, described O-tRNA is amber inhibition type tRNA, or O-tRNA comprises the polynucleotide sequence shown in SEQ ID NO:1.In these methods, provide the step of translation system also to comprise the nucleic acid that the coding target protein that contains described translation system amber selection codon used is provided.
Also can in host cell, implement to produce the method for the mutein that contains 3-imidazolyl tyrosine.In these situations, (the host cell providing comprises 3-imidazolyl tyrosine translation system of the present invention, nucleotide sequence, the O-tRNA sequence that comprises the O-RS that encodes and contain at least one and select the nucleic acid of coding target protein of codon), and under suitable culture condition, (for example, in substratum, add 3-imidazolyl tyrosine etc.) and cultivate this host cell special insertion 3-imidazolyl tyrosine that can cause fixing a point in described target protein.In some embodiments, provide step to comprise eubacterium host cell (for example, intestinal bacteria) is provided.
Thereby the present invention also provides and makes the myohaemoglobin mutant that production contains 3-imidazolyl tyrosine make myohaemoglobin produce the method for oxidase activity, described method is utilized above-mentioned 3-imidazolyl tyrosine translation system, wherein the nucleotide sequence of coding myohaemoglobin mutant used is SEQ ID NO:7, introduce 3-imidazolyl tyrosine for 33 at wild-type myohaemoglobin, 29 leucines are sported to Histidine, the aminoacid sequence of described myohaemoglobin mutant is SEQ ID NO:6 simultaneously.These methods start from the step that the 3-imidazolyl tyrosine translation system that contains following component is provided conventionally: (i) 3-imidazolyl tyrosine; (ii) orthogonal aminoacyl-tRNA synthetase (O-RS); (iii) orthogonal tRNA (O-tRNA), it comprises the polynucleotide sequence shown in SEQ ID NO:1, O-tRNA described in the preferential aminoacylation of described 3-imidazolyl tyrosine for wherein said O-RS; (iv) the encode nucleic acid of described myohaemoglobin, such as, but not limited to, SEQ ID NO:7, wherein said nucleic acid contain described O-tRNA specific recognition at least one select codon (being optionally amber codon); Then in the translation process of described protein; the O-tRNA of 3-imidazolyl tyrosine aminoacylation reacts to described selection codon and the described 3-imidazolyl tyrosine in substratum is fixed a point the selected location of the described myohaemoglobin of special insertion, to produce oxidase activity afterwards by introducing cupric ion in conjunction with center.
The present invention also provides the myohaemoglobin mutant that contains 3-imidazolyl tyrosine with oxidase activity that utilizes 3-imidazolyl tyrosine translation system of the present invention to produce, the aminoacid sequence of described myohaemoglobin mutant is SEQ ID NO:6, introduce 3-imidazolyl tyrosine for 33 at wild-type myohaemoglobin, 29 leucines are sported to Histidine, described myohaemoglobin mutant produces oxidase activity under suitable condition simultaneously.
In sum, the invention provides following:
1. orthogonal aminoacyl-tRNA synthetase, the group that its aminoacid sequence containing selects amino acid shown in free SEQ ID NO:2 and their examples of conservative variations to form.
2. a 3-imidazolyl tyrosine translation system, described system comprises:
(i) 3-imidazolyl tyrosine;
(ii) the orthogonal aminoacyl-tRNA synthetase described in the 1st;
(iii) orthogonal tRNA, it comprises the polynucleotide sequence shown in SEQ ID NO:1; Orthogonal tRNA described in the preferential aminoacylation of described 3-imidazolyl tyrosine for wherein said orthogonal aminoacyl-tRNA synthetase; With
(iv) nucleic acid of coding target protein, at least one selection codon that wherein said nucleic acid contains described orthogonal tRNA specific recognition.
3. the translation system as described in the 2nd, is characterized in that, described orthogonal tRNA is amber inhibition type tRNA, and described selection codon is amber codon.
4. the translation system as described in the 2nd, it also comprises the nucleotide sequence of coded orthogonal aminoacyl-tRNA synthetase.
5. a host cell, the nucleotide sequence that it comprises described orthogonal tRNA sequence and the described orthogonal aminoacyl-tRNA synthetase of coding.
6. the host cell as described in the 5th, wherein said host cell is eubacterium cell, preferably Bacillus coli cells.
7. a method that is created in the mutein of the special insertion of at least one selected location fixed point 3-imidazolyl tyrosine, described method comprises the steps:
(a) provide 3-imidazolyl tyrosine translation system claimed in claim 2, this system comprises:
(i) 3-imidazolyl tyrosine;
(ii) the orthogonal aminoacyl-tRNA synthetase described in the 1st;
(iii) orthogonal tRNA, it comprises the polynucleotide sequence shown in SEQ ID NO:1; Orthogonal tRNA described in the preferential aminoacylation of described 3-imidazolyl tyrosine for wherein said orthogonal aminoacyl-tRNA synthetase; With
(iv) the encode nucleic acid of described target protein, wherein said nucleic acid comprise described orthogonal tRNA specific recognition in selected position at least one select codon; With
(b) nucleic acid of the described target protein of coding is transformed in host cell claimed in claim 5; at the translate duration of described protein; the orthogonal tRNA of 3-imidazolyl tyrosine aminoacylation reacts to described selection codon and by the described selected location of the described target protein of special insertion of fixing a point of the 3-imidazolyl tyrosine in substratum, thereby is created in the described target protein of selected location containing 3-imidazolyl tyrosine.
8. the method as described in the 7th, wherein said orthogonal tRNA is amber inhibition type tRNA, and described selection codon is amber codon.
9. produce the method for the myohaemoglobin mutant that contains 3-imidazolyl tyrosine, it utilizes the method described in claim 7, wherein the nucleotide sequence of coding myohaemoglobin mutant used is SEQ ID NO:7, introduce 3-imidazolyl tyrosine for 33 at wild-type myohaemoglobin, 29 leucines are sported to Histidine, the aminoacid sequence of described myohaemoglobin mutant is SEQ ID NO:6 simultaneously.
10. the myohaemoglobin mutant that contains 3-imidazolyl tyrosine being obtained by the method described in the 9th, the aminoacid sequence of described myohaemoglobin mutant is SEQ ID NO:6, introduce 3-imidazolyl tyrosine for 33 at wild-type myohaemoglobin, 29 leucines are sported to Histidine, described myohaemoglobin mutant produces oxidase activity under suitable condition simultaneously.
3, beneficial effect
One of object of Protein Molecular Design is to disclose the biological principle that some cannot obtain by research native protein, and these new principles may have potential biological chemistry and the application prospect of biophysics.But, closely seeing some naturally occurring metalloprotein, we find that nature utilizes the kind of metal or its title complex very limited.And can to form the amino acid kind of coordination also very limited with these metals or its title complex, in 20 kinds of natural amino acids that nature exists, can form coordination less than half.For the limitation of natural coordination acidic amino acid, people are just attempting to be overcome by introduce alpha-non-natural amino acid in the process of the design & formulation of albumen.These alpha-non-natural amino acids and natural amino acid structurally have similarity, but its structure and properties is more diversified.
By the optionally modified protein of method of the orthogonal chemistry of biology, can realize protein loci specificity and insert alpha-non-natural amino acid.The application amber codon 3-imidazolyl tyrosine of encoding in cell, realizes specific site in myohaemoglobin and inserts this alpha-non-natural amino acid, for simulating the Tyr-His covalent cross-linking structure of HCO albumen; 29 contiguous leucines are mutated into Histidine by applied molecular biology method simultaneously, makes itself and 3-imidazolyl tyrosine, and 64 hyte propylhomoserins of far-end form cupric ion jointly in conjunction with center.After this mutain is combined with cupric ion, can be water molecules by oxygen conversion, and can produce active oxygen (ROS).In addition, also confirmed that by direct evidence Tyr-His covalent cross-linking structure plays vital effect to the oxidase catalyzed activity of HCO first herein.
Brief description of the drawings
In detailed description below in conjunction with accompanying drawing, above-mentioned feature and advantage of the present invention will be more obvious, wherein:
Fig. 1 is the chemosynthesis of (S)-2-amino-3-(4-hydroxyl-3-(1H-imidazoles-1-yl) phenyl) propionic acid (being abbreviated as imiTyr);
Fig. 2 is the nuclear magnetic spectrum of imiTyr;
Fig. 3 is orthogonal tRNA, aminoacyl-tRNA synthetase and Mb sequence;
Fig. 4 is that the SDS-PAGE electrophorogram of imiTyr-myohaemoglobin: A is the SDS-PAGE electrophorogram of imiTyr-Mb (4TAG), and B is the SDS-PAGE electrophorogram of imiTyrCuBMb;
Fig. 5 is the mass spectrum of imiTyr-Mb (4TAG);
Fig. 6 is the mass spectrum of imiTyrCuBMb;
Fig. 7 is that the ultraviolet-visible light spectrogram of Mb: A is the ultraviolet-visible light spectrogram of wild-type Mb, and B is the ultraviolet-visible light spectrogram of imiTyrCuBMb;
Fig. 8 is that in imiTyrCuBMb, titration adds Cu
2+ultraviolet-visible light spectrogram;
Fig. 9 is: A is that Mb and mutant thereof consume O
2the ability that produces water and ROS, B is imiTyrCuBMb and F33YCuBMb cycle consumption O
2ability.
Embodiment
Carry out by the following examples further to illustrate the present invention.But should be appreciated that, described embodiment is illustrational object, is not intended to limit scope and spirit of the present invention.
It should be appreciated by those skilled in the art that unless stated otherwise, in following embodiment, chemical reagent used is the reagent of the analytical pure rank that can buy by commercial sources.
The chemosynthesis (Fig. 1, Fig. 2) of embodiment 1:imiTyr
In 50ml three-necked bottle, add imidazoles (0.34g, 5mmol, purchased from sigma company), anhydrous Cs2CO3 (1.92g, 10mmol, purchased from Tianjin Alfa Aesar company), CuI (0.019g, 0.1mmol), Boc-L-3-iodotyrosine (Chinese name Boc-L-3-iodogorgonic acid) (2.03g, 5mmol, purchased from gill biochemical corp, Shanghai) and 8ml dry DMF (purchased from Beijing lark prestige company).Under the condition of nitrogen protection, stir and reflux 18 hours.After cooling, extract with ethyl acetate and distilled water, water carries out separation and purification (separator column YMC AA12S052503WT, purchased from Hui Deyi company, flow velocity 12ml/min) with preparation HPLC.Yield 50%.MS：m/z：248[M+H]+；1H-NMR(600MHz，DMSO-d6)：δ11.20(s，1H)，9.32(s，1H)，8.46(s，2H)，7.99(s，1H)，7.87(s，1H)，7.52(s，1H)，7.33(d，1H)，7.2(d，1H)，3.53(dd，1H)，3.19(m，2H)。
The required chemical reagent of above building-up reactions if no special instructions, all purchased from Beijing Chemical Plant, is the above rank of analytical pure.
Embodiment 2: evolution imiTyr specificity aminoacyl-tRNA synthetase
For locus specificity in gene inserts imiTyr; need in E.coli host cell used, introduce aminoacyl-tRNA synthetase/tRNA orthogonal right, this is orthogonal to deriving from Methanococcus jannaschii (Methanococcus jannaschii) amber inhibition tyrosyl tRNA (MjtRNACUA
tyr)/tyrosyl-t RNA synthetase (its aminoacid sequence is SEQ ID NO:5 for MjTyrRS, wild-type) is right.MjTyrRS mutation library is structured in card and receives in mycin resistance pBK plasmid (purchased from the Peter G.Schultz of scripps institute of U.S. laboratory), on this plasmid between the promotor and terminator of E.coli glutamine synthetase.The synthetic enzyme mutation library using is pBk-lib-jw1 storehouse, and the construction process of this mutation library is: on MjTyrRS gene, select 6 sites (Tyr32, Leu65, Phe108, Gln109, Asp158, and Leu162) introducing NNK sudden change (N=A+T+C+G; K=T+G), other 6 sites (Ile63, Ala67, His70, Tyr114, Ile159, Val164) or random mutation be Gly or remain unchanged (referring to Xie, J.; Liu, W.S.; Schultz, P.G.Angew.Chem., Int.Ed.2007,46,9239-9242; Wang, JY.; Zhang W.; Song WJ; Et al.J.Am.Chem.Soc.2010,132,14812-14818).
By the evolve aminoacyl-tRNA synthetase of specific recognition imiTyr of positive-negative selection.Just screen plasmid and comprising MjtRNA
cUA tyr, the chloramphenicol acetyl transferasegene of TAG sudden change, the t7 rna polymerase of the amber mutation of startup expressing green fluorescent protein, tetracycline resistance gene.Negative screening plasmid comprises MjtRNA
cUA tyr, the amber mutation barnase gene under arabinose operon, and ampicillin resistance gene.Carry out 3 and take turns positive-negative selection: include the E.coli DH10B cell that just screens plasmid as just screening host cell.Cell electricity turns pbk-lib-jw1 storehouse, SOC substratum (2% (W/V) Tryptones, 0.5% (W/V) yeast powder, 0.05% (W/V) NaCl, 2.5mM KCl, 10mM MgCl
2, 20mM glucose) cultivate 1 hour at 37 DEG C.Use afterwards the minimal medium (formula of GMML minimal medium: M9 salt/glycerine: 764g Na instead
2hPO
4.7H
2o or 30g Na
2hPO
4, 15g KH
2pO
4, 2.5g NaCl, 5g NH
4cl, 50ml glycerine, autoclaving, pH 7.0; 1M MgSO
4: autoclaving; 50mM CaCl
2: autoclaving; 25mM FeCl
2: filtration sterilization; 0.3M leucine: be dissolved in 0.3M NaOH filtration sterilization; 1L liquid GMML substratum: 200ml M9 salt/glycerine, 2ml MgSO
4, 2ml CaCl
2, 2ml FeCl
2, 1ml leucine) and wash twice, bed board solid minimal medium (adding 500ml 3% agar powder, 1mM imiTyr, 50mg/L kantlex, 60mg/L paraxin, 15mg/L tsiklomitsin in liquid GMML substratum), cultivates 60 hours for 37 DEG C.Collect cell, extract plasmid DNA, electrophoretic separation, glue reclaims.Then, will be transformed into through the pBK-lib-jw1 just screening in the DH10B competent cell that comprises negative screening plasmid.In SOC substratum, recover 1 hour.The LB solid medium (every liter of substratum is containing 10g Tryptones, 5g yeast powder, 10g NaCl) that coated plate comprises 0.2% pectinose (purchased from sigma company) afterwards.Cultivate 8-12 hour for 37 DEG C.Repeating altogether 3 takes turns.
Last is taken turns just to screen and chooses 384 clones, puts respectively plate and is containing 1mM imiTyr, paraxin 60,80,100, on the GMML solid medium of 120mg/L, and does not comprise imiTyr but comprises paraxin 0,20,40, the GMML solid medium of 60mg/L.Select on the substratum at 1mM imiTyr120mg/L paraxin and grow, and the clone who does not grow in 0mM imiTyr 40mg/L paraxin substratum further verifies.Choose 9 clones; wherein cloning 1 3-imidazolyl tyrosine inserts most effective; order-checking shows; the aminoacid sequence of clone's 1 aminoacyl-tRNA synthetase mutant (imiTyrRS) comprising is shown in SEQ ID NO:2; wherein mutational site is Tyr32Glu; Leu65Ser, His67Gly, Asp158Tyr and Leu162Asn.
Embodiment 3: express imiTyr-myohaemoglobin and Mass Spectrometric Identification
Orthogonal tRNA (SEQ ID NO:1) and the imiTyrRS (SEQ ID NO:2) that screens are building up to respectively to pEVOL carrier (purchased from the Peter G.Schultz of scripps institute of U.S. laboratory) upper, then cotransformation is to including in the DH10B cell (purchased from Quan Shi King Company) of pbad-myohaemoglobin (4TAG) (this plasmid is purchased from the Peter G.Schultz of scripps institute of U.S. laboratory) (wherein the nucleotides sequence of myohaemoglobin is classified SEQ ID NO:4 as).Picking is single is cloned in 37 DEG C of cultivations to OD
600approximate at 0.5 o'clock, in LB substratum, add 1mM imiTyr (purchased from gill biochemical corp, Shanghai), and 0.2% pectinose (purchased from sigma company) culturing cell, contrast does not add imiTyr.After 6-8 hour, receive bacterium, Ni-NTA purifying protein, and with SDS-PAGE electrophoretic analysis (Fig. 4 A).
We find, only in the substratum that has imiTyr, just can be purified into the myohaemoglobin of total length, and the imiTyrRS that this explanation screens can specific identification imiTyr.In LB substratum, the productive rate of imiTyr-myohaemoglobin is 10mg/L, and the productive rate of wild-type myohaemoglobin is 50mg/L.4 amber mutation sites that are only inserted into myohaemoglobin in order to detect imiTyr, we have carried out ESI-TOF mass spectrometric detection to imiTyr-myohaemoglobin, and detected result molecular weight is 18497Da (Fig. 5), coincide with the molecular weight 18496Da calculating.
Embodiment 4: express imiTyr-myohaemoglobin mutant as HCO functional mode
Can simulate the function of HCO in order to make myohaemoglobin, through the contrast of active sites, we have built myohaemoglobin double-mutant (nucleotide sequence is as shown in SEQ ID NO:7) by gene engineering method, wherein 29 leucines sport Histidine, 33 phenylalanines sport TAG terminator codon, then 33 special insertion imiTyr of fixed point at myohaemoglobin mutant by the same procedure in embodiment 3, express and produce imiTyrCuBMb mutain (aminoacid sequence is as shown in SEQ ID NO:6), and verify (Fig. 4 B with SDS-PAGE electrophoresis and ESI-TOF mass spectrum, Fig. 6).
After verifying Mb sudden change, whether albumen self conformation is exerted an influence, we have measured the ultraviolet-visible spectrum (Fig. 7) of imiTyrCuBMb under pH 7.4 conditions, find that it is similar to wild-type Mb, there is strong absorption peak Soret band at 408nm place.When the V-Brite B with excessive (being commonly called as vat powder) is reduced into ferric iron-imiTyrCuBMb after deoxidation-imiTyrCuBMb, measure its ultraviolet-visible spectrum, find that it has respectively Soret band and visible absorption band at 430nm and 562nm place, this and deoxidation-wtMb are also on all four.Above result shows to introduce in mutant imiTyrCuBMb the conformational change that does not cause albumen after alpha-non-natural amino acid.
Due to protein mutation and introduce after alpha-non-natural amino acid, its 29 hyte propylhomoserin, 64 hyte propylhomoserins of 33 3-imidazolyl tyrosine and far-end can form cupric ion binding site jointly.We add the Cu of different concns under pH 7.4 conditions toward titration in imiTyrCuBMb
2+, measure ultraviolet-visible spectrum (Fig. 8).Result shows, Soret is with along with Cu
2+the variation of concentration constantly reduces, and finally stable, this means in imiTyrCuBMb and has successfully introduced a cupric ion binding site, according to double reciprocal plot and Hill figure, measuring result analysis is shown its K
dvalue is 1.6 μ M.
Because the function of HCO is mainly catalysis O
2form water and do not discharge active oxygen (ROS), therefore we use oxygen electrode (Oxygraph Clark-type oxygen electrode, Hansatech Instruments) at 25 DEG C, in the 20mM Tris damping fluid of pH 7.4, measure imiTyrCuBMb (6 μ M) catalysis O
2the ability of reduction, with the vitamins C (6mM) of 1000 equivalents, as reductive agent, the TMPD of 100 equivalents (N ', N '-tetramethyl-para-phenylene diamine dihydrochloride, 0.6mM, purchased from Beijing lark prestige company for N, N) is as electronics transmission intermediate.Meanwhile, in order to verify whether imiTyrCuBMb can catalysis O
2form active oxygen (ROS), we select catalase (catalase, 7.3U/ μ L, purchased from sigma company) and superoxide-dismutase (SOD, 0.5U/ μ L, purchased from sigma company) to monitor respectively H
2o
2formation with superoxide.If O
2consumption be non-formation water owing to having formed ROS, so along with the adding of catalase and SOD, O
2reduction ratio can slow down gradually, because these two kinds of enzymes can be by H
2o
2and peroxide breakdown, again form O
2.Measuring result is as shown in Figure 9 A: 1, no matter whether add Cu
2+(6 μ M CuSO4), wild-type Mb can consume most of O
2and form ROS; 2, at equivalent Cu
2+under the condition existing, imiTyrCuBMb can catalysis O
2form water, only produce the H lower than 6%
2o
2; If 3 do not add Cu
2+, imiTyrCuBMb can be by 30% O
2change into ROS.This result shows, Cu
bin catalytic process, play an important role.
Except Cu
b, we infer that Tyr-His covalent cross-linking also has important effect.In order to confirm this inference, we have built mutant Leu29His/Phe33Tyr/Phe43His (F33YCuBMb), wherein, 29,43 and 64 His can form a cupric ion binding site, and in site on Mb of 33Tyr and 43His and structure and imiTyrCuBMb the imidazole group of 33 imiTyr and phenolic group group similar, just do not form covalent cross-linking.Compare imiTyrCuBMb, F33YCuB-Mb is by 50% O
2change into ROS (Fig. 9 A), confirmed that Tyr-His covalent cross-linking is at selective catalysis O
2in the process of formation water, also there is vital effect.
In order to test imiTyrCuBMb catalytic reduction O under cycling condition
2ability, we add 6 μ M imiTyrCuBMb, 6 μ M CuSO4,800 μ M O
2, 0.6mM TMPD and 6mM vitamins C, monitor O with oxygen electrode
2expenditure Levels.Work as O
2consume completely, then add subsequently 800 μ M O
2, continue to monitor with oxygen electrode.So repeat, after 1000 circulations, to find O
2wear rate almost do not weaken, show that the catalysis of imiTyrCuBMb shows no sign of decline.By contrast, F33YCuBMb can only carry out 400 circulations (Fig. 9 B) under the same conditions.This result has further confirmed that Tyr-His covalent cross-linking is at selective catalysis O
2form the vital role in water process.
Should be appreciated that, although with reference to its exemplary embodiment, the present invention is shown particularly and described, but will be understood by those skilled in the art that, under the condition not deviating from by the defined the spirit and scope of the present invention of accompanying claim, the variation of various forms and details can be carried out therein, the arbitrary combination of various embodiments can be carried out.
Claims (11)
1. orthogonal aminoacyl-tRNA synthetase, its aminoacid sequence is SEQ ID NO:2.
2. a 3-imidazolyl tyrosine translation system, described system comprises:
(i) 3-imidazolyl tyrosine;
(ii) orthogonal aminoacyl-tRNA synthetase claimed in claim 1;
(iii) orthogonal tRNA, it is the polynucleotide sequence shown in SEQ ID NO:1; Orthogonal tRNA described in the preferential aminoacylation of described 3-imidazolyl tyrosine for wherein said orthogonal aminoacyl-tRNA synthetase; With
(iv) nucleic acid of coding target protein, at least one selection codon that wherein said nucleic acid contains described orthogonal tRNA specific recognition.
3. translation system as claimed in claim 2, is characterized in that, described orthogonal tRNA is amber inhibition type tRNA, and described selection codon is amber codon.
4. translation system as claimed in claim 2, it also comprises the nucleotide sequence of the described orthogonal aminoacyl-tRNA synthetase of encoding.
5. a host cell, the nucleotide sequence that it comprises the orthogonal tRNA sequence shown in SEQ ID NO:1 and coding orthogonal aminoacyl-tRNA synthetase claimed in claim 1.
6. host cell as claimed in claim 5, wherein said host cell is eubacterium cell.
7. host cell as claimed in claim 6, wherein said eubacterium cell is Bacillus coli cells.
8. a method that is created in the mutein of the special insertion of at least one selected location fixed point 3-imidazolyl tyrosine, described method comprises the steps:
(a) provide 3-imidazolyl tyrosine translation system claimed in claim 2, this system comprises:
(i) 3-imidazolyl tyrosine;
(ii) orthogonal aminoacyl-tRNA synthetase claimed in claim 1;
(iii) orthogonal tRNA, it is the polynucleotide sequence shown in SEQ ID NO:1; Orthogonal tRNA described in the preferential aminoacylation of described 3-imidazolyl tyrosine for wherein said orthogonal aminoacyl-tRNA synthetase; With
(iv) the encode nucleic acid of described target protein, wherein said nucleic acid comprise described orthogonal tRNA specific recognition in selected position at least one select codon; With
(b) nucleic acid of the described target protein of coding is transformed in host cell claimed in claim 5; at the translate duration of described protein; the orthogonal tRNA of 3-imidazolyl tyrosine aminoacylation reacts to described selection codon and by the described selected location of the described target protein of special insertion of fixing a point of the 3-imidazolyl tyrosine in substratum, thereby is created in the described target protein of selected location containing 3-imidazolyl tyrosine.
9. method as claimed in claim 8, wherein said orthogonal tRNA is amber inhibition type tRNA, and described selection codon is amber codon.
10. produce the method for the myohaemoglobin mutant that contains 3-imidazolyl tyrosine, it utilizes the method described in claim 8, wherein the nucleotide sequence of coding myohaemoglobin mutant used is SEQ IDNO:7, introduce 3-imidazolyl tyrosine for 33 at wild-type myohaemoglobin, 29 leucines are sported to Histidine, the aminoacid sequence of described myohaemoglobin mutant is SEQ ID NO:6 simultaneously.
The 11. myohaemoglobin mutant that contain 3-imidazolyl tyrosine that obtained by method claimed in claim 10, the aminoacid sequence of described myohaemoglobin mutant is SEQ ID NO:6, introduce 3-imidazolyl tyrosine for 33 at wild-type myohaemoglobin, 29 leucines are sported to Histidine, described myohaemoglobin mutant produces oxidase activity under suitable condition simultaneously.
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