CN103215235A - 3-imidazolyl tyrosine translation system and use thereof - Google Patents
3-imidazolyl tyrosine translation system and use thereof Download PDFInfo
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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 the aminoacyl-tRNA synthetase mutant, its aminoacid sequence that contains is selected from the group that is made of amino acid shown in the SEQ ID NO:2 and their examples of conservative variations.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 quadrature tRNA, quadrature aminoacyl-tRNA synthetase and their pairing with 3-imidazolyl tyrosine fix a point special insertion target protein 3-imidazolyl tyrosine translation system and utilize in target protein, the fix a point method of special insertion 3-imidazolyl tyrosine of described translation system.The invention still further relates to the mutein that contains 3-imidazolyl tyrosine that produces with this translation system and this method, for example, contain the myohaemoglobin mutant of 3-imidazolyl tyrosine, and the application that contains the mutein of 3-imidazolyl tyrosine.
Background technology
Protein is the essential substance basis of all life entities and vital movement thereof.In whole albumen, it all is some metallic albumen that 1/3rd albumen is arranged approximately, is called metalloprotein (Metalloprotein) or metalloenzyme (Metalloenzyme).Reduced hematin (Hemoproteins) be exactly wherein a big class contain protoporphyrin IX (protoheme, heme) as the metalloprotein of prothetic group, in the 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, and bio-sensing function (CO transmitter CooA and NO transmitter sGC etc.) CcP etc.).Though reduced hematin has widely different biological function, they have the protein molecular of almost similar protoheme prothetic group and similar protein and peptide chain parcel protoheme and form mode.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 long-term and extensive studies, attempt to answer this problem, and by the answer of this problem being familiar with proteic structure-character-reaction-function (structure-property-reactivity-function, the exquisite relation that is contained between SPRF).
Protoheme-copper oxydase (HCOs) is the dual combination center protein that existing protoheme has copper again, is that a big class extensively is present in the terminal oxidase on eucaryon plastosome and the bacterial respiratory chain.This proteinoid can be accepted four electronics from substrate, and is delivered on the oxygen molecule, oxygen finally is converted into two water moleculess, and can produce active oxygen (ROS) in this process.Wherein protoheme in conjunction with the center can with an axial Histidine coordination, and copper in conjunction with center (CuB) can with three contiguous Histidine coordinations, in the heart cupric ion and iron ion is apart from pact in two
Scientists is mainly passed through structure biology at present, zymetology, spectrum, methods such as x-ray and plan ecological model come structure and the function of HCO are studied, and obtained huge breakthrough, but still existed the associated problem needs of some important structure and mechanism aspects further to inquire into.For example, CuB in conjunction with and activate to play what effect aspect the oxygen actually? at protoheme-copper in conjunction with the center, how does the reduction of oxygen concentration follow the transmission or the picked-up of proton? 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 at present also Nobody Knows about the definite function of this structure.
In order further to understand CuB and the function of Tyr-His covalent cross-linking in HCO, there is the people to simulate HCO albumen by in yeast cell pigment c oxydase and Physter macrocephalus myohaemoglobin, introducing the copper center.Myohaemoglobin (Myoglobin wherein, Mb) form by 153 amino acid and a protoheme prothetic group, have little, the stable height of molecular weight, easily obtain characteristics such as high purity protein by genetically engineered and protein engineering, for a long time always extremely investigator's parent look at, extensively the model as protein molecule is used for studying particularly reduced hematin 26S Proteasome Structure and Function relation of protein.The biological function of Mb is to store and the transportation 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 hole of the 6th axial existence, can be reversibly in conjunction with O
2Molecule.Simultaneously, two conservative amino-acid residue His64 and Val68 can stablize O around the hole
2The existence of molecule.At present, people progressively recognize, the difference of reduced hematin biological function depends primarily on the structural difference in protoheme active centre, comprise the coordination situation of protoheme center iron, the microenvironment of protoheme binding cavity, and the mode of action between protoheme and the protein peptide chain or the like.For the difference on intermolecular structure of these naturally occurring reduced hematins and the function, be not to be insurmountable barrier.Growing along with genetically engineered and protein engineering, now, people can carry out the design and the structure of protein molecular reasoningly, by the active centre structure of reduced hematin is done suitably to modify and adjustment, 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 the mutual conversion between the dissimilar reduced hematin or the like.
Introducing other metal ion binding site in the reduced hematin molecule, is a bright spot in reduced hematin structure and the function study on the transformation field.By Mb and the oxidasic active centre of protoheme-copper structure are done careful comparison, amino acid Leu29 and Phe43 in the hole, Mb active centre are mutated into His, originally the far-end part His64 that exists in the hole in addition, in resulting mutant Mb L29H/F43H molecule, just there are 3 His in its active centre, and they can be in conjunction with a Cu
2+Ion (being called CuBMb).Existing investigator is through experiment confirm, this mutant since lack a proton and an electronics pipeline and at O
2In conjunction with can making heme degraded in the reduction process, thereby also reflect the importance of the covalent cross-linking of Tyr-His among protoheme-copper oxydase HCO from the side.3-imidazolyl tyrosine has the characteristic group of Tyr-His covalent cross-linking, and promptly imidazolyl and 3 carbon of tyrosine is covalently bound.In order further to study these proteinic 26S Proteasome Structure and Functions, this area also needs the proteinic new departure of special insertion of this non-natural 3-imidazolyl tyrosine being fixed a point.Now developed in protokaryon and eukaryote locus specificity ground fixed point in the various alpha-non-natural amino acid bodies has been inserted proteinic universal method.These methods depend on quadrature protein translation component, and described component discerns suitable selection codon (selector codon) thereby the polypeptide translate duration inserts the qualification position with required alpha-non-natural amino acid in vivo.These methods are utilized the quadrature tRNA (O-tRNA) of identification selection codon, and corresponding specificity quadrature aminoacyl-tRNA synthetase (O-RS) loads this O-tRNA with alpha-non-natural amino acid.These components not with the intravital any endogenous tRNA of host living beings, aminoacyl-tRNA synthetase (RS), amino acid or codon cross reaction (that is, it must be orthogonal).Utilize the different alpha-non-natural amino acid of this quadrature tRNA-RS pairing possibility a large amount of structures of genetic coding.
This area is generally known to utilize and is suitable for preparing the proteinic orthogonal translation system that contains one or more alpha-non-natural amino acids, for example produces 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, and its denomination of invention is " IN VIVO INCORPORATION OF UNNATURAL AMINO ACIDS "; WO 2004/094593, and its denomination of invention is " EXPANDING THE EUKARYOTIC GENETIC CODE ".Other of the orthogonal translation system of special insertion alpha-non-natural amino acid of fixing a point and their generation and using method are discussed 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 the aminoacyl-tRNA synthetase mutant, its aminoacid sequence that contains is selected from the group that is made of amino acid shown in the SEQ ID NO:2 and their examples of conservative variations.The present invention relates to utilize quadrature tRNA, quadrature aminoacyl-tRNA synthetase and their pairing with 3-imidazolyl tyrosine fix a point special insertion target protein 3-imidazolyl tyrosine translation system and utilize in target protein, the fix a point method of special insertion 3-imidazolyl tyrosine of described translation system.The invention still further relates to the mutein and the application thereof that contain 3-imidazolyl tyrosine that produce with this translation system and this method.
Therefore; the object of the present invention is to provide and utilize quadrature tRNA, quadrature aminoacyl-tRNA synthetase and their pairing the 3-imidazolyl tyrosine proteinic 3-imidazolyl of the special insertion tyrosine translation system of fixing a point, and the method that provides this translation system in target protein, to fix a point special insertion 3-imidazolyl tyrosine.
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, the inventor utilizes this method that 3-imidazolyl tyrosine is fixed a point in the special insertion myohaemoglobin (Myoglobin), and this albumen can produce oxidase activity by introducing cupric ion in conjunction with the center.Yet, it should be appreciated by those skilled in the art that method of the present invention also can be used for the special insertion 3-imidazolyl tyrosine of multiple protein fixed point outside myohaemoglobin, is not limited to this albumen.
2, technical scheme
The inventor obtains a kind of quadrature aminoacyl-tRNA synthetase through screening, and its aminoacid sequence that contains is selected from the group that is made of amino acid shown in the SEQ ID NO:2 and their examples of conservative variations.And the inventor utilizes described quadrature aminoacyl-tRNA synthetase, has researched and developed 3-imidazolyl tyrosine translation system.
Specifically, the invention provides in vivo (for example in host cell) alpha-non-natural amino acid 3-imidazolyl tyrosine fixed a point to selecting codon (selector codon) to react 3-imidazolyl tyrosine translation system of the polypeptide chain of special insertion in extending as amber terminator codon (TAG).Described 3-imidazolyl tyrosine translation system does not comprise matches with interactional quadrature-tRNA of host cell translating mechanism (O-tRNA) and quadrature aminoacyl-tRNA synthetase (O-RS).That is, host cell endogenous aminoacyl-tRNA synthetase is not used amino acid (natural or non-natural) and is loaded O-tRNA.Similarly, but O-RS provided by the invention not not load endogenous tRNA with amino acid (natural or non-natural) under conspicuous level or some situation with detection level ground.Utilize described translation system to produce to contain a large amount of protein of the special insertion of fixed point 3-imidazolyl tyrosine 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) quadrature aminoacyl-tRNA synthetic enzyme (O-RS); (c) quadrature tRNA (O-tRNA); it comprises the polynucleotide sequence shown in the SEQ ID NO:1; wherein said quadrature aminoacyl-tRNA synthetic enzyme is with described alpha-non-natural amino acid (being 3-imidazolyl tyrosine), the preferential described O-tRNA of 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 quadrature 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.
Used quadrature aminoacyl-tRNA synthetase (O-RS) is the aminoacyl tRNA synthetase mutant that the inventor finds in the described system, and its aminoacid sequence that contains is selected from the group that is made of amino acid shown in the SEQ ID NO:2 and their examples of conservative variations.
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) quadrature aminoacyl-tRNA synthetase;
(iii) quadrature tRNA, it comprises the polynucleotide sequence shown in the SEQ ID NO:1; Wherein said quadrature aminoacyl-tRNA synthetase is with the described quadrature tRNA of the preferential aminoacylation of described 3-imidazolyl tyrosine; With
(iv) the encode nucleic acid of target protein, wherein said nucleic acid contain described quadrature tRNA specific recognition at least one select codon.
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, each component in this translation system is derived from Methanococcus jannaschii (Methanococcus jannaschii).For example, quadrature tRNA (O-tRNA) sports and amber codon complementary tyrosine tRNA for the anticodon in ancient bacterium source.In some embodiments, O-tRNA is amber inhibition type tRNA.In some embodiments, O-tRNA comprises the polynucleotide sequence shown in the SEQ ID NO:1, and preferably, the sequence of O-tRNA is shown in SEQ ID NO:1.In one embodiment, the quadrature aminoacyl-tRNA synthetase (O-RS) that is used for this system can comprise the aminoacid sequence shown in the SEQ ID NO:2 and the conservative variant of this sequence.
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 quadrature tRNA (O-tRNA) specific recognition.In aspect preferred, described quadrature tRNA is amber inhibition type tRNA, and described selection codon is an amber codon.
In certain aspects, the invention provides the host cell of the nucleotide sequence that comprises quadrature tRNA sequence and coded orthogonal aminoacyl-tRNA synthetase.Used host cell is not done concrete qualification, as long as O-RS and O-tRNA keep their orthogonality in their host cell environment.For example, described host cell can be the eubacterium cell, as intestinal bacteria.As described in embodiment, can be in host cell with the recombinant vectors cotransformation of nucleotide sequence that comprises the recombinant vectors of quadrature tRNA sequence and comprise coded orthogonal 3-imidazolyl tyrosine aminoacyl-tRNA synthetase, and obtain to comprise the host cell of the nucleotide sequence of quadrature 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 usually: (i) alpha-non-natural amino acid, i.e. 3-imidazolyl tyrosine; (ii) quadrature aminoacyl-tRNA synthetase (O-RS); (iii) quadrature tRNA (O-tRNA), it comprises the polynucleotide sequence shown in the SEQ ID NO:1, and wherein said O-RS is with the preferential described O-tRNA of aminoacylation of described alpha-non-natural amino acid (being 3-imidazolyl tyrosine); (iv) the encode nucleic acid of target protein, wherein said nucleic acid contain the O-tRNA specific recognition at least one select codon (randomly being amber codon); The nucleic acid of described target protein of will encoding then is transformed in the host cell of the nucleotide sequence that comprises quadrature tRNA sequence and coded orthogonal aminoacyl-tRNA synthetase; in described proteinic translation process; the O-tRNA of 3-imidazolyl tyrosine aminoacylation reacts to described selection codon and with the selected location of the described target protein of special insertion of fixing a point of the 3-imidazolyl tyrosine in the substratum, thereby is created in the mutein that 3-imidazolyl tyrosine is contained in the selected location.The host cell that wherein comprises the nucleotide sequence of quadrature tRNA sequence and coded orthogonal 3-imidazolyl tyrosine aminoacyl-tRNA synthetase can obtain in selected host cell by recombinant vectors that will comprise quadrature tRNA sequence and the recombinant vectors cotransformation that comprises the nucleotide sequence of coded orthogonal 3-imidazolyl tyrosine aminoacyl-tRNA synthetase.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 aminoacyl-tRNA synthetase mutant (that is the used quadrature aminoacyl-tRNA synthetase of the present invention) with the preferential described O-tRNA of 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 that obtains 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 amber codon complementary tyrosine tRNA, for example, described O-tRNA is amber inhibition type tRNA, and perhaps O-tRNA comprises the polynucleotide sequence shown in the 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 the used amber selection codon of described translation system 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 that provides comprises 3-imidazolyl tyrosine translation system of the present invention (promptly, comprise nucleotide sequence, the O-tRNA sequence of the O-RS that encodes and contain the nucleic acid that at least one selects the coding target protein of codon), can cause the special insertion 3-imidazolyl tyrosine of fixed point in described target protein and under the suitable culture condition, (for example, in substratum, add 3-imidazolyl tyrosine etc.) and cultivate this host cell.In some embodiments, provide step to comprise eubacterium host cell (for example, intestinal bacteria) is provided.
Thereby the present invention also provides the method that the myohaemoglobin mutant that contains 3-imidazolyl tyrosine makes myohaemoglobin generation oxidase activity of producing that makes, described method is utilized above-mentioned 3-imidazolyl tyrosine translation system, the nucleotide sequence of wherein used coding myohaemoglobin mutant is SEQ ID NO:7, introduce 3-imidazolyl tyrosine for 33 at the wild-type myohaemoglobin, simultaneously 29 leucines are sported Histidine, the aminoacid sequence of described myohaemoglobin mutant is SEQ ID NO:6.These methods start from the step that the 3-imidazolyl tyrosine translation system that contains following component is provided usually: (i) 3-imidazolyl tyrosine; (ii) quadrature aminoacyl-tRNA synthetase (O-RS); (iii) quadrature tRNA (O-tRNA), it comprises the polynucleotide sequence shown in the SEQ ID NO:1, and wherein said O-RS is with the described O-tRNA of the described preferential aminoacylation of 3-imidazolyl tyrosine; (iv) the encode nucleic acid of described myohaemoglobin for example, but is not limited to, SEQ ID NO:7, wherein said nucleic acid contain described O-tRNA specific recognition at least one select codon (randomly being amber codon); Then in described proteinic translation process; the O-tRNA of 3-imidazolyl tyrosine aminoacylation reacts to described selection codon and the described 3-imidazolyl tyrosine in the substratum is fixed a point the selected location of the described myohaemoglobin of special insertion to produce oxidase activity by introducing cupric ion in conjunction with the center afterwards.
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 the wild-type myohaemoglobin, simultaneously 29 leucines are sported Histidine, described myohaemoglobin mutant produces oxidase activity under suitable condition.
In sum, the invention provides following:
1. quadrature aminoacyl-tRNA synthetase, its aminoacid sequence that contains are selected from the group that is made of amino acid shown in the SEQ ID NO:2 and their examples of conservative variations.
2. 3-imidazolyl tyrosine translation system, described system comprises:
(i) 3-imidazolyl tyrosine;
(ii) the 1st described quadrature aminoacyl-tRNA synthetase;
(iii) quadrature tRNA, it comprises the polynucleotide sequence shown in the SEQ ID NO:1; Wherein said quadrature aminoacyl-tRNA synthetase is with the described quadrature tRNA of the preferential aminoacylation of described 3-imidazolyl tyrosine; With
(iv) the encode nucleic acid of target protein, wherein said nucleic acid contain described quadrature tRNA specific recognition at least one select codon.
3. as the 2nd described translation system, it is characterized in that described quadrature tRNA is amber inhibition type tRNA, and described selection codon is an amber codon.
4. as the 2nd described translation system, it also comprises the nucleotide sequence of coded orthogonal aminoacyl-tRNA synthetase.
5. host cell, it comprises the nucleotide sequence of described quadrature tRNA sequence and the described quadrature aminoacyl-tRNA synthetase of coding.
6. as the 5th described host cell, wherein said host cell is the eubacterium cell, preferred Bacillus coli cells.
7. 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 the described 3-imidazolyl of claim 2 tyrosine translation system, this system comprises:
(i) 3-imidazolyl tyrosine;
(ii) the 1st described quadrature aminoacyl-tRNA synthetase;
(iii) quadrature tRNA, it comprises the polynucleotide sequence shown in the SEQ ID NO:1; Wherein said quadrature aminoacyl-tRNA synthetase is with the described quadrature tRNA of the preferential aminoacylation of described 3-imidazolyl tyrosine; With
(iv) the encode nucleic acid of described target protein, wherein said nucleic acid comprise described quadrature tRNA specific recognition in selected position at least one select codon; With
(b) will the encode nucleic acid of described target protein is transformed in the described host cell of claim 5; at described proteinic translate duration; the quadrature tRNA of 3-imidazolyl tyrosine aminoacylation reacts to described selection codon and with the described selected location of the described target protein of special insertion of fixing a point of the 3-imidazolyl tyrosine in the substratum, thereby is created in the described target protein that the selected location contains 3-imidazolyl tyrosine.
8. as the 7th described method, wherein said quadrature tRNA is amber inhibition type tRNA, and described selection codon is an amber codon.
9. produce the method for the myohaemoglobin mutant that contains 3-imidazolyl tyrosine, it utilizes the described method of claim 7, the nucleotide sequence of wherein used coding myohaemoglobin mutant is SEQ ID NO:7, introduce 3-imidazolyl tyrosine for 33 at the wild-type myohaemoglobin, simultaneously 29 leucines are sported Histidine, the aminoacid sequence of described myohaemoglobin mutant is SEQ ID NO:6.
10. the myohaemoglobin mutant that contains 3-imidazolyl tyrosine that obtains by the 9th described method, the aminoacid sequence of described myohaemoglobin mutant is SEQ ID NO:6, introduce 3-imidazolyl tyrosine for 33 at the wild-type myohaemoglobin, simultaneously 29 leucines are sported Histidine, described myohaemoglobin mutant produces oxidase activity under suitable condition.
3, beneficial effect
One of purpose of protein molecule design be disclose some by the research native protein the biological principle that can't obtain, and these new principles may have the application prospect of potential biological chemistry and biophysics.Yet, 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, can form in 20 kinds of natural amino acids that nature exists 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 proteic design and structure.These alpha-non-natural amino acids and natural amino acid structurally have similarity, but its structure and properties is more diversified.
Method by biological quadrature chemistry is modified protein optionally, can realize that the protein loci specificity inserts alpha-non-natural amino acid.Use the amber codon 3-imidazolyl tyrosine of in cell, encoding, be implemented in that specific site inserts this alpha-non-natural amino acid in the myohaemoglobin, be used for simulating the Tyr-His covalent cross-linking structure of HCO albumen; 29 leucines that while applied molecular biology method will be close to are mutated into Histidine, make itself and 3-imidazolyl tyrosine, and 64 hyte propylhomoserins of far-end form cupric ion jointly in conjunction with the center.When this mutain with after cupric ion combines, can be water molecules with oxygen conversion, and can not produce active oxygen (ROS).In addition, this paper has also confirmed that with direct evidence Tyr-His covalent cross-linking structure plays crucial effects to the oxidase catalyzed activity of HCO first.
Description of drawings
From the 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 quadrature 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 titration adds Cu among the imiTyrCuBMb
2+The ultraviolet-visible light spectrogram;
Fig. 9 is: A is that Mb and mutant thereof consume O
2Produce the ability of water and ROS, B is imiTyrCuBMb and F33YCuBMb cycle consumption O
2Ability.
Embodiment
Come further to illustrate the present invention by the following examples.But should be appreciated that described embodiment is illustrational purpose, and be not intended to limit the scope of the invention and spirit.
It should be appreciated by those skilled in the art that unless stated otherwise used chemical reagent is other reagent of analytical pure level that can buy by commercial sources among the following embodiment.
The chemosynthesis of embodiment 1:imiTyr (Fig. 1, Fig. 2)
In the 50ml three-necked bottle, add imidazoles (0.34g, 5mmol, available from sigma company), anhydrous Cs2CO3 (1.92g, 10mmol, available from Tianjin Alfa Aesar company), CuI (0.019g, 0.1mmol), Boc-L-3-iodotyrosine (Chinese name Boc-L-3-iodogorgonic acid) (2.03g, 5mmol is available from Shanghai gill biochemical corp) and 8ml dry DMF (available from Beijing lark prestige company).Under the condition of nitrogen protection, stir and refluxed 18 hours.After the cooling, extract with ethyl acetate and distilled water, water carries out separation and purification (separator column YMC AA12S052503WT is available from intelligent De Yi 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 all available from the Beijing Chemical Plant, is the above rank of analytical pure if no special instructions.
Embodiment 2: evolution imiTyr specificity aminoacyl-tRNA synthetase
For locus specificity in gene inserts imiTyr; it is right to introduce aminoacyl-tRNA synthetase/tRNA quadrature in used E.coli host cell, and this quadrature suppresses tyrosyl tRNA (MjtRNACUA to deriving from Methanococcus jannaschii (Methanococcus jannaschii) amber
Tyr)/tyrosyl-t RNA synthetase (its aminoacid sequence is SEQ ID NO:5 for MjTyrRS, wild-type) is right.MjTyrRS sudden change storehouse is structured in card and receives in the mycin resistance pBK plasmid (available from U.S. scripps institute Peter G.Schultz laboratory), between the promotor and terminator of E.coli glutamine synthetase on this plasmid.Employed synthetic enzyme sudden change storehouse is the pBk-lib-jw1 storehouse, and the construction process in this sudden change storehouse is: select 6 sites (Tyr32, Leu65, Phe108, Gln109, Asp158, and Leu162) and introduce NNK sudden change (N=A+T+C+G on the MjTyrRS gene; 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 conduct of just screening plasmid and just screening host cell.The cell electricity changes 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) cultivated 1 hour at 37 ℃.Use the minimal medium (prescription of GMML minimal medium: M9 salt/glycerine: 764g Na afterwards 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 among the 0.3M NaOH filtration sterilization; 1L liquid GMML substratum: 200ml M9 salt/glycerine, 2ml MgSO
4, 2ml CaCl
2, 2ml FeCl
2, the 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) was cultivated 60 hours for 37 ℃.Collect cell, extract plasmid DNA, electrophoretic separation, glue reclaims.Then, will be transformed into through the pBK-lib-jw1 that is just screening in the DH10B competent cell that comprises negative screening plasmid.Recovered 1 hour in the SOC substratum.Coated plate comprises the LB solid medium (every liter of substratum contains the 10g Tryptones, 5g yeast powder, 10g NaCl) of 0.2% pectinose (available from sigma company) afterwards.Cultivated 8-12 hour for 37 ℃.Repeating 3 altogether takes turns.
Last is taken turns just to screen and chooses 384 clones, puts plate respectively 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 and on the substratum of 1mM imiTyr120mg/L paraxin, growing, 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) that is comprised is shown in the SEQ ID NO:2; wherein the mutational site is Tyr32Glu; Leu65Ser, His67Gly, Asp158Tyr and Leu162Asn.
Embodiment 3: express imiTyr-myohaemoglobin and mass spectrum and identify
Quadrature tRNA (SEQ ID NO:1) and the imiTyrRS (SEQ ID NO:2) that screens are building up to respectively on the pEVOL carrier (available from U.S. scripps institute Peter G.Schultz laboratory), and cotransformation is in the DH10B cell that includes pbad-myohaemoglobin (4TAG) (this plasmid is available from U.S. scripps institute Peter G.Schultz laboratory) (wherein the nucleotides sequence of myohaemoglobin is classified SEQ ID NO:4 as) (available from full formula King Company) then.Picking is single to be cloned in 37 ℃ and to cultivate OD
600Approximate at 0.5 o'clock, in the LB substratum, add 1mM imiTyr (available from Shanghai gill biochemical corp), and 0.2% pectinose (available from sigma company) culturing cell, contrast does not add imiTyr.After 6-8 hour, receive bacterium, the Ni-NTA purifying protein, and with SDS-PAGE electrophoretic analysis (Fig. 4 A).
We find, only just can be purified into the myohaemoglobin of total length in having the substratum of imiTyr, and the imiTyrRS that this explanation screens can specific identification imiTyr.The productive rate of imiTyr-myohaemoglobin is 10mg/L in the LB substratum, and the productive rate of wild-type myohaemoglobin is 50mg/L.In order to detect 4 amber mutation sites that imiTyr only is inserted into myohaemoglobin, we have carried out the ESI-TOF mass spectrometric detection to the imiTyr-myohaemoglobin, and the detected result molecular weight is 18497Da (Fig. 5), coincide with the molecular weight 18496Da that calculates.
Embodiment 4: express imiTyr-myohaemoglobin mutant as the HCO functional mode
Can simulate the function of HCO in order to make myohaemoglobin, contrast through the active centre structure, we have made up myohaemoglobin double-mutant (nucleotide sequence is shown in SEQ ID NO:7) with gene engineering method, wherein 29 leucines sport Histidine, 33 phenylalanines sport the TAG terminator codon, then with 33 the fixed point special insertion imiTyrs of the same procedure among the embodiment 3 at the myohaemoglobin mutant, express and produce imiTyrCuBMb mutain (aminoacid sequence is shown in SEQ ID NO:6), and with SDS-PAGE electrophoresis and ESI-TOF mass spectrum verify (Fig. 4 B, Fig. 6).
Whether albumen self conformation is exerted an influence after verifying the Mb sudden change, we, finds that it is similar to wild-type Mb, have strong absorption peak Soret band at the 408nm place if having measured the ultraviolet-visible spectrum (Fig. 7) of imiTyrCuBMb under pH 7.4 conditions.After ferric iron-imiTyrCuBMb being reduced into deoxidation-imiTyrCuBMb with excessive V-Brite B (being commonly called as vat powder), measure its ultraviolet-visible spectrum, find that it has Soret band and visible absorption band respectively at 430nm and 562nm place, this and deoxidation-wtMb also are on all four.Above result shows among the mutant imiTyrCuBMb and not to cause proteic conformational change after introducing alpha-non-natural amino acid.
Since protein mutation and introduce alpha-non-natural amino acid after, its 29 hyte propylhomoserin, 64 hyte propylhomoserins of 33 3-imidazolyl tyrosine and far-end can form the cupric ion binding site jointly.We under pH 7.4 conditions in the imiTyrCuBMb titration add the Cu of different concns
2+, measure ultraviolet-visible spectrum (Fig. 8).The result shows that Soret is with along with Cu
2+The variation of concentration constantly reduces, and final stable, this means and has successfully introduced a cupric ion binding site among the imiTyrCuBMb, according to double reciprocal plot and Hill figure measuring result is carried out analysis revealed, its K
DValue is 1.6 μ M.
Because the function of HCO mainly is catalysis O
2Form water and do not discharge active oxygen (ROS), therefore we use oxygen electrode (Oxygraph Clark-type oxygen electrode, Hansatech Instruments), measured imiTyrCuBMb (6 μ M) catalysis O in the 20mM Tris damping fluid of pH 7.4 at 25 ℃
2The reductive ability, as reductive agent, 100 normal TMPD (N ', N '-tetramethyl-para-phenylene diamine dihydrochloride, 0.6mM is available from Beijing lark prestige company for N, N) are as the electron transport intermediate with 1000 normal vitamins Cs (6mM).Simultaneously, in order to verify whether imiTyrCuBMb can catalysis O
2Form active oxygen (ROS), we select for use catalase (catalase, 7.3U/ μ L is available from sigma company) and superoxide-dismutase (SOD, 0.5U/ μ L is available from sigma company) to monitor H respectively
2O
2Formation with superoxide.If O
2Consumption be owing to formed ROS but not form water, so along with the adding of catalase and SOD, O
2Reduction ratio can slow down gradually, because these two kinds of enzymes can be with H
2O
2And peroxide breakdown, form O again
2Measuring result is shown in Fig. 9 A: 1, no matter whether add Cu
2+(6 μ M CuSO4), wild-type Mb can consume most of O
2And formation ROS; 2, at equivalent Cu
2+Under the condition that exists, but imiTyrCuBMb catalysis O
2Form water, only generation is lower than 6% H
2O
23, if do not add Cu
2+, imiTyrCuBMb can be with 30% O
2Change into ROS.This result shows, Cu
BIn catalytic process, play an important role.
Except Cu
B, we infer that the Tyr-His covalent cross-linking also has important effect.In order to confirm this inference, we have made up mutant Leu29His/Phe33Tyr/Phe43His (F33YCuBMb), wherein, 29,43 and 64 His can form a cupric ion binding site, and 33Tyr and 43His on Mb the site and structure and imiTyrCuBMb in the imidazole group of 33 imiTyr and phenolic group group similar, just do not form covalent cross-linking.Compare imiTyrCuBMb, F33YCuB-Mb is with 50% O
2Changed into ROS (Fig. 9 A), confirmed that the Tyr-His covalent cross-linking is at selection catalysis O
2In the process of formation water crucial effects is arranged also.
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 are monitored O with oxygen electrode
2The consumption situation.Work as O
2Consume fully, then add 800 μ M O subsequently
2, continue to monitor with oxygen electrode.After so repeating 1000 circulations, 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 confirmed that further the Tyr-His covalent cross-linking is at selection catalysis O
2Form the vital role in the water process.
Should be appreciated that, although with reference to its exemplary embodiment, the present invention is shown particularly and describe, but will be understood by those skilled in the art that, under the condition that does not deviate 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 (10)
1. quadrature aminoacyl-tRNA synthetase, its aminoacid sequence that contains are selected from the group that is made of amino acid shown in the SEQ ID NO:2 and their examples of conservative variations.
2. 3-imidazolyl tyrosine translation system, described system comprises:
(i) 3-imidazolyl tyrosine;
The (ii) described quadrature aminoacyl-tRNA synthetase of claim 1;
(iii) quadrature tRNA, it comprises the polynucleotide sequence shown in the SEQ ID NO:1; Wherein said quadrature aminoacyl-tRNA synthetase is with the described quadrature tRNA of the preferential aminoacylation of described 3-imidazolyl tyrosine; With
(iv) the encode nucleic acid of target protein, wherein said nucleic acid contain described quadrature tRNA specific recognition at least one select codon.
3. translation system as claimed in claim 2 is characterized in that, described quadrature tRNA is amber inhibition type tRNA, and described selection codon is an amber codon.
4. translation system as claimed in claim 2, it also comprises the nucleotide sequence of coded orthogonal aminoacyl-tRNA synthetase.
5. host cell, it comprises the nucleotide sequence of described quadrature tRNA sequence and the described quadrature aminoacyl-tRNA synthetase of coding.
6. host cell as claimed in claim 5, wherein said host cell are the eubacterium cells, preferred Bacillus coli cells.
7. 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 the described 3-imidazolyl of claim 2 tyrosine translation system, this system comprises:
(i) 3-imidazolyl tyrosine;
The (ii) described quadrature aminoacyl-tRNA synthetase of claim 1;
(iii) quadrature tRNA, it comprises the polynucleotide sequence shown in the SEQ ID NO:1; Wherein said quadrature aminoacyl-tRNA synthetase is with the described quadrature tRNA of the preferential aminoacylation of described 3-imidazolyl tyrosine; With
(iv) the encode nucleic acid of described target protein, wherein said nucleic acid comprise described quadrature tRNA specific recognition in selected position at least one select codon; With
(b) will the encode nucleic acid of described target protein is transformed in the described host cell of claim 5; at described proteinic translate duration; the quadrature tRNA of 3-imidazolyl tyrosine aminoacylation reacts to described selection codon and with the described selected location of the described target protein of special insertion of fixing a point of the 3-imidazolyl tyrosine in the substratum, thereby is created in the described target protein that the selected location contains 3-imidazolyl tyrosine.
8. method as claimed in claim 7, wherein said quadrature tRNA is amber inhibition type tRNA, and described selection codon is an amber codon.
9. produce the method for the myohaemoglobin mutant that contains 3-imidazolyl tyrosine, it utilizes the described method of claim 7, the nucleotide sequence of wherein used coding myohaemoglobin mutant is SEQ ID NO:7, introduce 3-imidazolyl tyrosine for 33 at the wild-type myohaemoglobin, simultaneously 29 leucines are sported Histidine, the aminoacid sequence of described myohaemoglobin mutant is SEQ ID NO:6.
10. the myohaemoglobin mutant that contains 3-imidazolyl tyrosine that obtains by the described method of claim 9, the aminoacid sequence of described myohaemoglobin mutant is SEQ ID NO:6, introduce 3-imidazolyl tyrosine for 33 at the wild-type myohaemoglobin, simultaneously 29 leucines are sported Histidine, described myohaemoglobin mutant produces oxidase activity under suitable condition.
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| CN105985944A (en) * | 2015-01-29 | 2016-10-05 | 中国科学院生物物理研究所 | Novel method of intracellular site-specific covalent RNA labeling |
| CN105985944B (en) * | 2015-01-29 | 2019-05-28 | 中国科学院生物物理研究所 | A kind of new method of intracellular site specific covalent labeled RNA |
| CN104894091A (en) * | 2015-06-19 | 2015-09-09 | 北京理工大学 | Method of improving enzymatic thermostability via artificially designed glycosylation modification |
| CN104894091B (en) * | 2015-06-19 | 2018-08-21 | 北京理工大学 | A kind of glycosyl modified method for improving enzyme heat stability of engineer |
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