CN104004723B - 3,5-bis-fluoro tyrosine translation system and application thereof - Google Patents
3,5-bis-fluoro tyrosine translation system and application thereof Download PDFInfo
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- CN104004723B CN104004723B CN201310056306.4A CN201310056306A CN104004723B CN 104004723 B CN104004723 B CN 104004723B CN 201310056306 A CN201310056306 A CN 201310056306A CN 104004723 B CN104004723 B CN 104004723B
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Abstract
The present invention relates to a kind of aminoacyl tRNA synthetase mutant; it is a kind of orthogonal aminoacyl tRNA synzyme; the aminoacid sequence that it contains selects the group of the examples of conservative variations composition of the aminoacid sequence shown in free SEQ ID NO:4 and the aminoacid sequence shown in SEQ ID NO:4, and described examples of conservative variations has the enzymatic activity identical with the aminoacid sequence shown in SEQ ID NO:4.The present invention also provides for a kind of 3,5 two fluoro tyrosine translation system, and it comprises: (i) 3,5 two fluorotyrosine；(ii) the orthogonal aminoacyl tRNA synzyme of the present invention；(iii) orthogonal tRNA, wherein said orthogonal aminoacyl tRNA synzyme orthogonal tRNA described in described 3, the 5 two preferential aminoacylations of fluorotyrosine；(iv) nucleic acid of encoding target protein, wherein said nucleic acid contains at least one selection codon of described orthogonal tRNA specific recognition.
Description
Technical field
The invention belongs to biochemical field.Specifically, the present invention provides a kind of aminoacyl-tRNA synthetase mutant,
It is a kind of orthogonal aminoacyl-tRNA synzyme, the aminoacid sequence choosing aminoacid shown in free SEQ ID NO:4 that it contains
The group of the examples of conservative variations composition of the aminoacid sequence shown in sequence and SEQ ID NO:4, described examples of conservative variations has and SEQ
The enzymatic activity that aminoacid sequence shown in ID NO:4 is identical.The invention still further relates to a kind of 3,5-bis-fluorotyrosine (is abbreviated as
F2Y) translation system.More particularly it relates to utilize orthogonal tRNA, orthogonal aminoacyl-tRNA synzyme pairing by 3,
5-bis-fluorotyrosine fixed point specificity inserts the 3 of target protein, 5-bis-fluoro tyrosine translation system, and turns over described in utilization
System of translating pinpoints specificity in target protein and inserts 3, the method for 5-bis-fluorotyrosine.The invention still further relates to turn over this set
Translate that system and this method produce containing 3, the mutein of 5-bis-fluorotyrosine, such as, insert 3,5-bis-fluoro cheese
The protokaryon protein tyrosine kinase mutant of propylhomoserin, and insert 3, the application of the mutein of 5-bis-fluorotyrosine.
Background technology
Protein phosphorylation is the most necessary for the initiation of many biological phenomenons, including cell growth, propagation, ubiquitin
(ubiquitin) processes such as the protein degradation mediated.Particularly tyrosine phosphorylation, adjusts as cell signalling and enzymatic activity
A kind of major way of control, generally by causing the interaction between protein, and then the mediating growth factor, hormone and thin
Intracellular cytokines etc. are to the signals-modulating of receptor on cell membrane.But, tyrosine phosphorylation is institute in all phosphorylation modifications of cell
The ratio accounted for is the lowest.The cell protein of general 10% can be by phosphorylation covalent modification, but the phosphorylation of every 100 albumen
Modification only has the modification of 1 tyrosine group.Compared with the serine in most cells and threonine phosphorylation level, cheese
The horizontal estimated of propylhomoserin phosphorylation is low 2000 times.Just because of in cell tyrosine phosphorylation be on close level low, Cai Nengbao
Card cell, under the stimulation of internal/external signal, makes sensitive reaction, so the phosphorylation of research tyrosine is for cell signal
The research of regulation and control and many important biomolecule phenomenons has a particularly important meaning, and to the protein of tyrosine phosphorylation occurs
Identify and the action site to the regulation and control and medicine that disclose cell processes of identifying of phosphorylation site plays very important work
With.
19F-NMR technology is owing to its chemical shift range is big, be difficult to peak overlapping and sensitivity advantages of higher occur, from last
Since the fifties in century occurs, in analyzing the research such as optical isomer, life sciences, there is its Special Significance.Nuclear-magnetism is altogether in vivo
Shake in technology,19F-NMR is compared with additive method, under conditions of not lesioned sample, so that it may carry out near real-time quantitative monitoring.Nearly 30
Year, introduce tetraalkylammonium fluoride and carry out internal19The research of F-NMR, the research of fluorine-containing bioactive substance and fluorine material is subject to
The biggest attention,19F-NMR technology and application development thereof get up.From the point of view of the situation of nearly 10 years, grinding of fluorine-containing bioactive substance
Studying carefully and apply by the biggest attention at medicine, pesticide and studying of the fluorine material with excellent properties, this is recent19F-
One main feature of NMR research and development.
According to previous report, containing 3, the peptide fragment of 5-bis-fluorotyrosine as the substrate of protein tyrosine kinase, its
Show the efficiency being similar to as substrate with the corresponding peptide fragment containing tyrosine, and be not significantly affected by protein-tyrosine
The catalysis activity of phosphatase.Therefore this research is intended to be extended by genetic code, with alpha-non-natural amino acid 3,5-bis-fluoro cheese ammonia
Acid substitutes tyrosine so that it is as tetraalkylammonium fluoride, such that it is able to pass through19F-NMR technology detects and quantifies tyrosine phosphatase
Change level.Meanwhile, this research developed site-specific in various alpha-non-natural amino acid bodies in protokaryon and eukaryote
Property ground fixed point insert protein universal method.These methods depend on orthogonal protein translation component, and described component identification is closed
Suitable selection codon (selector codon) is it is thus possible to insert required alpha-non-natural amino acid during polypeptide translation in vivo
Enter defined position.These methods utilize the orthogonal tRNA (O-tRNA) of identification selection codon, and the corresponding orthogonal ammonia of specificity
Acyl group-tRNA synzyme (O-RS) loads this O-tRNA with alpha-non-natural amino acid.These components not with in host living beings body appoint
What endogenous tRNA, aminoacyl-tRNA synthetase (RS), aminoacid or codon cross reaction (that is, it must be orthogonal).
Utilize the alpha-non-natural amino acid of the possible a large amount of configurations of genetic coding of this orthogonal tRNA-RS pairing.
Utilize as commonly known in the art and be suitable for preparation and turn over containing the orthogonal of protein of one or more alpha-non-natural amino acids
Translate system, such as, produce the universal method of orthogonal translation system.For example, with reference to international publication number WO 2002/086075, its
Bright entitled " METHODS AND COMPOSITION FOR THE PRODUCTION OF ORTHOGONAL tRNA-
AMINOACYL-tRNA SYNTHETASE PAIRS”；WO 2002/085923, its invention entitled " IN VIVO
INCORPORATION OF UNNATURAL AMINO ACIDS”；WO 2004/094593, its invention entitled " EXPANDING
THE EUKARYOTIC GENETIC CODE”.Fixed point specificity insert the orthogonal translation system of alpha-non-natural amino acid and they
Generation and using method other discuss referring also 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,
Methods36 (3): 227-238 (2005)；Xie and Schultz, Curr.Opinion in Chemical Biology9 (6):
548-554(2005)；Wang etc., Annu.Rev.Biophys.Biomol.Struct.35:225-249 (2006).
Summary of the invention
1, technical problem
The present invention provides a kind of aminoacyl-tRNA synthetase mutant, and it is a kind of orthogonal aminoacyl-tRNA synzyme,
Aminoacid sequence that it contains selects aminoacid shown in free SEQ ID NO:4 and the aminoacid sequence shown in SEQ ID NO:4
The group of examples of conservative variations composition, described examples of conservative variations has the enzyme identical with the aminoacid sequence shown in SEQ ID NO:4 and lives
Property.This aminoacyl-tRNA synthetase mutant can be with 3, and the preferential aminoacylation of 5-bis-fluorotyrosine (being abbreviated as F2Y) is therewith
The orthogonal tRNA of pairing, thus in the aminoacid sequence of translation, insert F2Y.This is that the present inventor finds first, meanwhile, and this
Inventor have also resolved its high-resolution crystal structure, correspondingly, in the present invention by its named orthogonal 3,5-bis-fluoro
Tyrosine aminoacyl-tRNA synthetase (F2YRS).
On the basis of above-mentioned discovery, the present invention provides a kind of and utilizes orthogonal tRNA, orthogonal aminoacyl-tRNA synzyme
Pairing is by 3, and 5-bis-fluorotyrosine fixed point specificity inserts the 3 of target protein, 5-bis-fluoro tyrosine translation system, and profit
In target protein, pinpoint specificity by described translation system and insert 3, the method for 5-bis-fluorotyrosine.The invention still further relates to
With this translation system and this method produce containing 3, the mutein of 5-bis-fluorotyrosine and application thereof.
Therefore, it is an object of the invention to provide utilize orthogonal tRNA, orthogonal aminoacyl-tRNA synzyme pairing by 3,
5-bis-fluorotyrosine fixed point specificity inserts the 3 of protein, 5-bis-fluoro tyrosine translation system, and provides and utilize this to turn over
System of translating pinpoints specificity in target protein and inserts 3, the method for 5-bis-fluorotyrosine.
The present invention also provides for utilizing the 3 of the present invention, 5-bis-fluoro tyrosine translation system produce containing at least one 3,
The mutein of 5-bis-fluorotyrosine.In preferred aspects of the invention, the present inventor's profit in this way by 3,5-bis-
Fluorotyrosine fixed point specificity inserts in destination protein, and described destination protein includes, but not limited to protokaryon protein-tyrosine
Kinases Etk.3 are comprised, the protokaryon protein tyrosine kinase mutant of 5-bis-fluorotyrosine by what the method for the present invention obtained
Albumen as tetraalkylammonium fluoride, thus can pass through19F-NMR technology detects and quantifies tyrosine phosphorylation level.But, this
Skilled person is it should be understood that the method for the present invention can be used for determining in the multiple protein outside protein tyrosine kinase
Point specificity inserts 3,5-bis-fluorotyrosine, it is not limited to this albumen.2, technical scheme
The present inventor is through screening, it is thus achieved that a kind of orthogonal aminoacyl-tRNA synthase mutant, and it is a kind of Orthogonal aminoacyl
Base-tRNA synzyme, the aminoacid sequence that it contains selects aminoacid sequence and SEQ ID NO:4 shown in free SEQ ID NO:4
The group of the examples of conservative variations composition of shown aminoacid sequence, described examples of conservative variations has and the ammonia shown in SEQ ID NO:4
The enzymatic activity that base acid sequence is identical, in the present invention by its named orthogonal 3,5-bis-fluorotyrosine aminoacyl-tRNA synthesize
Enzyme (F2YRS).Meanwhile, the present inventor have also resolved its high-resolution crystal structure.Further, the present inventor utilizes described orthogonal
Aminoacyl-tRNA synthetase, have developed 3,5-bis-fluoro tyrosine translation system.
It should be appreciated by those skilled in the art that in the present invention, except the aminoacid sequence shown in SEQ ID NO:4 it
Outward, term " orthogonal aminoacyl-tRNA synzyme of the present invention " or " orthogonal 3,5-bis-fluorotyrosine aminoacyl-tRNA synthesis
Enzyme " also include the examples of conservative variations of aminoacid sequence shown in SEQ ID NO:4, as long as described examples of conservative variations has and SEQ ID
The enzymatic activity that aminoacid sequence shown in NO:4 is identical；And also include the aminoacid sequence shown in SEQ ID NO:4
Through one or more amino acid whose replacements, lack or add and have identical with the aminoacid sequence shown in SEQ ID NO:4
The aminoacid sequence derivative by the aminoacid sequence shown in SEQ ID NO:4 of enzymatic activity.
Further, orthogonal the 3 of code book invention, the nucleotide of 5-bis-fluorotyrosine aminoacyl-tRNA synthetase (F2YRS)
Sequence is intended to be included within the scope of the present invention.Preferably, described coding nucleotide sequence is shown in SEQ ID NO:3.
Specifically, the present invention provides identification selection codon (selector (such as in host cell) in vivo
Codon) such as Amber stop codon (TAG) thus by alpha-non-natural amino acid 3,5-bis-fluorotyrosine fixed point specificity is inserted into
In polypeptide chain 3,5-bis-fluoro tyrosine translation system.Described 3,5-bis-fluoro tyrosine translation system comprise thinless with host
Orthogonal-tRNA (O-tRNA) and orthogonal aminoacyl-tRNA synzyme (O-RS) that born of the same parents' translating mechanism interacts match.That is, place
Chief cell endogenous aminoacyl-tRNA synthetase will not identify O-tRNA.Similarly, the O-RS that the present invention provides is not with notable water
Put down or do not identify endogenous tRNA with detectable level.Utilize described translation system can produce in translation
During pinpoint specificity insert 3, a large amount of protein of 5-bis-fluorotyrosine.
In certain aspects, the present invention provides 3,5-bis-fluoro tyrosine translation system.Described translation system comprises:
(a) alpha-non-natural amino acid, i.e. 3,5-bis-fluorotyrosine,
The Orthogonal aminoacyl-tRNA synzyme (O-RS) of (b) present invention, and
C () orthogonal tRNA (O-tRNA), it comprises the polynucleotide sequence shown in SEQ ID NO:1, wherein said orthogonal
Aminoacyl-tRNA synthetase with described alpha-non-natural amino acid (i.e. 3,5-bis-fluorotyrosine), O-tRNA described in preferential aminoacylation.
Preferably, the 3 of the present invention, 5-bis-fluoro tyrosine translation system also comprises the nucleic acid of encoding target protein, its
Described in nucleic acid contain by orthogonal tRNA (O-tRNA) specific recognition at least one select codon, it is therefore preferable to succinum is close
Numeral.It is highly preferred that the 3 of the present invention, 5-bis-fluoro tyrosine translation system also comprises coded orthogonal aminoacyl-tRNA synthetase
Nucleotide sequence.
Orthogonal aminoacyl-tRNA synzyme (O-RS) used in described system is the aminoacyl that the present inventor finds first
Base tRNA synthase mutant, the aminoacid sequence that it contains selects aminoacid sequence and SEQ ID shown in free SEQ ID NO:4
Aminoacid sequence shown in NO:4 examples of conservative variations composition group, described examples of conservative variations have with shown in SEQ ID NO:4
The identical enzymatic activity of aminoacid sequence.
In preferred aspects of the invention, the present invention provides one 3,5-bis-fluoro tyrosine translation system, described system
Comprise:
(i) 3,5-bis-fluorotyrosine；
(ii) orthogonal aminoacyl-tRNA synzyme of the present invention；
(iii) orthogonal tRNA, it comprises the polynucleotide sequence shown in SEQ ID NO:1；Wherein said orthogonal aminoacyl-
TRNA synzyme with described 3, orthogonal tRNA described in the preferential aminoacylation of 5-bis-fluorotyrosine；With
(iv) nucleic acid of encoding target protein, wherein said nucleic acid contains described orthogonal tRNA specific recognition at least
One selection codon.
Preferably, described 3,5-bis-fluoro tyrosine translation system also comprise code book invention orthogonal aminoacyl-tRNA
The nucleotide sequence of synzyme.
Various components in this translation system can be derived from various source of species, such as, and each group in this translation system
Divide derived from Methanococcus jannaschii (Methanococcus jannaschii).Such as, orthogonal tRNA (O-tRNA) is that ancient bacterium comes
The anticodon in source sports the tyrosine tRNA complementary with amber codon.In some embodiments, O-tRNA is that succinum presses down
Type tRNA processed.In some embodiments, O-tRNA comprises the polynucleotide sequence shown in SEQ ID NO:1, it is preferable that O-
The sequence of tRNA is as shown in SEQ ID NO:1.In one embodiment, the orthogonal aminoacyl-tRNA for this system synthesizes
Enzyme can comprise the aminoacid sequence shown in SEQ ID NO:4 and the conservative variant of this sequence.In preferred embodiments, use
Aminoacid sequence in the orthogonal aminoacyl-tRNA synzyme of this system is shown in SEQ ID NO:4.
In certain aspects, the 3 of the present invention, 5-bis-fluoro tyrosine translation system also comprises the core of encoding target protein
Acid, wherein said nucleic acid has at least one by orthogonal tRNA (O-tRNA) specific recognition and selects codon.In preferably side
In face, described orthogonal tRNA is amber suppression tRNA, and described selection codon is amber codon.
In certain aspects, the present invention provides the nucleotide of the orthogonal aminoacyl-tRNA synzyme comprising code book invention
Sequence and the host cell of corresponding orthogonal tRNA sequence.Host cell used is not especially limited, if Orthogonal aminoacyl
Base-tRNA synzyme and orthogonal tRNA retain their orthogonality in their host cell environment.Such as, described place
Chief cell can be eubacteria cell, preferably escherichia coli.
The present invention also provides for producing and inserts 3 at least one selected location fixed point specificity, dashing forward of 5-bis-fluorotyrosine
Become method of protein.Described method utilizes above-mentioned 3,5-bis-fluoro tyrosine translation system.Described method generally includes following
Step:
A () provides 3 containing following components, the step of 5-bis-fluoro tyrosine translation system:
(i) alpha-non-natural amino acid, i.e. 3,5-bis-fluorotyrosine；
(ii) orthogonal aminoacyl-tRNA synzyme (O-RS) of the present invention；
(iii) orthogonal tRNA (O-tRNA), it comprises the polynucleotide sequence shown in SEQ ID NO:1, wherein said O-
RS with 3, O-tRNA described in the preferential aminoacylation of 5-bis-fluorotyrosine；With
(iv) nucleic acid of encoding target protein, wherein said nucleic acid contains at least one choosing of O-tRNA specific recognition
Select codon (being optionally amber codon)；
(b) by described orthogonal tRNA sequence and encode described orthogonal aminoacyl-tRNA synzyme nucleotide sequence and
The nucleotide sequence encoding described target protein clones and is transformed in suitable host cell, adds 3 in the medium, 5-bis-
Fluorotyrosine, in the translation process of described target protein, the orthogonal RNA of 3,5-bis-fluorotyrosine aminoacylations identifies and compiles
Selection codon and 3 on the mRNA of the described target protein of code, 5-bis-fluorotyrosine, thus mediate 3,5-bis-fluoro cheese
Propylhomoserin fixed point specificity inserts the amino acid position that described selection codon is corresponding, thus produces and contain 3 in selected location, 5-bis-
The mutein of fluorotyrosine.
It should be appreciated by those skilled in the art that the structure of suitable recombinant vector and the screening of host cell often can be passed through
Rule molecule clone technology and triage techniques realize.
It should be appreciated by those skilled in the art that in step (b), by described orthogonal tRNA sequence and the described orthogonal ammonia of coding
The nucleotide sequence of acyl group-tRNA synzyme and encode the nucleotide sequence of described target protein clone and be transformed into suitable
Host cell can be carried out in several ways, such as, by described orthogonal tRNA sequence, encode described orthogonal aminoacyl-
The nucleotide sequence of tRNA synzyme and encode the nucleotide sequence of described target protein and be operatively connected to respectively fit
When carrier in, then with in any order or three's cotransformation to suitable host cell；Or, it is also possible to by described orthogonal
TRNA sequence and encode the nucleotide sequence of described orthogonal aminoacyl-tRNA synzyme be operatively connected to one suitable
Carrier (connects with or without suitable joint between two kinds of sequences), by operable for the nucleotide sequence encoding described target protein
Property be connected in another kind of different suitable carrier, then by two kinds of recombinant vector cotransformation building to suitable
In host cell；Or, it is also possible to by operable for the nucleotide sequence of described orthogonal tRNA sequence and the described target protein of coding
Property be connected in a suitable carrier (connecting with or without suitable joint between two kinds of sequences), described orthogonal ammonia to be encoded
The nucleotide sequence of acyl group-tRNA synzyme is operatively connected in another kind of different suitable carrier, then by structure
The two kinds of recombinant vector cotransformation built up are in suitable host cell.Or, it is also possible to by orthogonal tRNA sequence and coding
The nucleotide sequence of described orthogonal aminoacyl-tRNA synzyme and encoding target nucleic acid sequences to proteins are with the most suitable
Order is operatively connected to together, is then cloned on a carrier, is finally transformed in suitable host cell.Above-mentioned
Cloning approach is all feasible, and those skilled in the art can experimental need easily to carry out suitable selection.
It addition, it should also be realized by those skilled in the art that in order to avoid " play and remove " to external source recombinant vector of host cell
Effect, often selecting to build with the carrier with different antibiotic markers needs cotransformation in same host cell
Nucleic acid sequence fragments.Selection, the structure of recombinant vector, the conversion of host cell or transfection for suitable carrier etc., all
It is the ordinary skill in the art, for example, it is possible to see the molecular cloning handbook that Cold Spring Harbor Laboratory is published.
In some embodiments of described method, it is provided that the step of translation system includes making wild type by direct mutagenesis
The amino acid binding pocket of aminoacyl-tRNA synthetase is undergone mutation, and selects with described alpha-non-natural amino acid (i.e. 3,5-bis-fluoro
Tyrosine) aminoacyl-tRNA synthetase mutant (that is, the Orthogonal aminoacyl used by the present invention of O-tRNA described in preferential aminoacylation
Base-tRNA synzyme).Described selection step is carried out from the aminoacyl-tRNA synthetase molecular library obtained after including direct mutagenesis
The just selection of described O-RS and negative selection (seeing following embodiment 2).In some embodiments, it is provided that the step of translation system
Also including the sequence providing O-tRNA, O-tRNA is that the anticodon in ancient bacterium source sports the tyrosine complementary with amber codon
TRNA, such as, described O-tRNA is amber suppression tRNA, or O-tRNA comprises the polynucleotide shown in SEQ ID NO:1
Sequence.In these methods, it is provided that the step of translation system also includes providing selecting containing the succinum used by described translation system
The nucleic acid of the encoding target protein of codon.
Also can implement to produce containing 3 in host cell, the method for the mutein of 5-bis-fluorotyrosine.At these
In situation, it is provided that host cell comprise the 3 of the present invention, 5-bis-fluoro tyrosine translation system (that is, comprises code book invention
The nucleotide sequence of O-RS, O-tRNA sequence and the nucleic acid of the encoding target protein containing at least one selection codon), and
Under suitable condition of culture, (such as, add 3 in the medium, 5-bis-fluorotyrosine etc.) cultivate this host cell and may result in
In described target protein, pinpoint specificity insert 3,5-bis-fluorotyrosine.In some embodiments, it is provided that step bag
Offer eubacterial host cell (such as, escherichia coli) is provided.
The present invention also provides for producing containing 3, the method for the tyrosine-kinase enzyme mutant of 5-bis-fluorotyrosine, in its utilization
State and produce at least one selected location fixed point specificity insertion 3, the method for the mutein of 5-bis-fluorotyrosine, wherein
It is special that the nucleotide sequence of coding protokaryon protein tyrosine kinase mutant used comprises described orthogonal tRNA in selected position
Property identification selection codon, during the translation of tyrosine kinase, 3,5-bis-fluorotyrosines fixed point be inserted into described selection
The amino acid position that codon is corresponding, thus produce and contain 3 at select location, the tyrosine-kinase enzyme mutant of 5-bis-fluorotyrosine
Body.
Preferably, the present invention also provides for producing containing 3, the protokaryon protein tyrosine kinase mutant of 5-bis-fluorotyrosine
Method, described method utilizes above-mentioned 3, and 5-bis-fluoro tyrosine translation system is carried out, and described method generally includes following step
Rapid:
A () provides 3 containing following components, the step of 5-bis-fluoro tyrosine translation system:
(i) 3,5-bis-fluorotyrosine；
(ii) orthogonal aminoacyl-tRNA synzyme (O-RS)；
(iii) orthogonal tRNA (O-tRNA), it comprises the polynucleotide sequence shown in SEQ ID NO:1, wherein said O-
RS with described 3, O-tRNA described in the preferential aminoacylation of 5-bis-fluorotyrosine；With
(iv) nucleic acid of described protokaryon protein tyrosine kinase is encoded, such as, but not limited to, SEQ ID NO:7, Qi Zhongsuo
State at least one selection codon (being optionally amber codon) that nucleic acid contains described O-tRNA specific recognition；
(b) by described orthogonal tRNA sequence and encode described orthogonal aminoacyl-tRNA synzyme nucleotide sequence and
The nucleotide sequence encoding described target protein clones and is transformed in suitable host cell, adds 3 in the medium, 5-bis-
Fluorotyrosine, in the translation process of described target protein (i.e. protokaryon protein tyrosine kinase), 3,5-bis-fluoro cheese ammonia
Selection codon and 3 on the mRNA of the orthogonal RNA recognition coding tyrosine kinase of acid aminoacylation, 5-bis-fluorotyrosine,
Thus mediating 3,5-bis-fluorotyrosine fixed point inserts ad-hoc location (that is, the described selection codon pair of described target protein
The amino acid position answered).
The present invention also provides for utilizing the 3 of the present invention, 5-bis-fluoro tyrosine translation system produce as tetraalkylammonium fluoride
Containing 3, the protokaryon protein tyrosine kinase mutant of 5-bis-fluorotyrosine, at wild type protokaryon protein tyrosine kinase
574 introduce 3, and 5-bis-fluorotyrosine, the aminoacid sequence of described tyrosine-kinase enzyme mutant is SEQ ID NO:8.Described
Kinase mutants can carry out the autophosphorylation of active centre tyrosine as wild type tyrosine kinases under optimum conditions,
Thus activate the protein kinase activity of self.
The present invention also provides for the method for the Tyr phosphorylation site identifying target protein of a kind of novelty, described method bag
Including and utilize the 3 of the present invention, presumption in described target protein is carried out the tyrosine of phosphorylation by 5-bis-fluoro tyrosine translation system
It is substituted by 3,5-bis-fluorotyrosine, thus obtain containing 3, the target protein mutant of 5-bis-fluorotyrosine, utilize this mesh
Mark protein mutant, as tetraalkylammonium fluoride, passes through19F-NMR technology detects 3, the phosphorylation of 5-bis-fluorotyrosine, thus
Identify whether described tyrosine site is Tyr phosphorylation site.
For example, as it is known that protokaryon protein tyrosine kinase has the autophosphorylation ability of lower degree, the most do not close
Suitable technology can detect and quantify himself phosphorylation level.But, utilizing the 3 of the present invention, 5-bis-fluorotyrosine turns over
574, protokaryon protein tyrosine kinase activity center tyrosine residue is replaced with 3 by system of translating, and 5-bis-fluorotyrosine obtains
Containing 3, the protokaryon protein tyrosine kinase mutant of 5-bis-fluorotyrosine, then pass through19F-NMR technology detects and quantifies
Self tyrosine phosphorylation level of this protokaryon protein tyrosine kinase mutant.This protokaryon protein tyrosine kinase mutant
Tyrosine phosphorylation level can represent the autophosphorylation level of wild type protokaryon protein tyrosine kinase to a certain extent.
Additionally, this testing result also demonstrates that 574, protokaryon protein tyrosine kinase activity center tyrosine is himself tyrosine phosphorus
Polyadenylation sites.
Therefore, the present invention provides following:
1. an orthogonal aminoacyl-tRNA synzyme, the aminoacid sequence that it contains selects ammonia shown in free SEQ ID NO:4
The group of the examples of conservative variations composition of the aminoacid sequence shown in base acid sequence and SEQ ID NO:4, described examples of conservative variations has
The enzymatic activity identical with the aminoacid sequence shown in SEQ ID NO:4.
2. one kind 3,5-bis-fluoro tyrosine translation system, described system comprises:
(i) 3,5-bis-fluorotyrosine；
(ii) the 1st described orthogonal aminoacyl-tRNA synzyme；
(iii) orthogonal tRNA, it comprises the polynucleotide sequence shown in SEQ ID NO:1；Wherein said orthogonal aminoacyl-
TRNA synzyme with described 3, orthogonal tRNA described in the preferential aminoacylation of 5-bis-fluorotyrosine；With
(iv) nucleic acid of encoding target protein, wherein said nucleic acid contains described orthogonal tRNA specific recognition at least
One selection codon.
3. the translation system as described in the 2nd, it is characterised in that described orthogonal tRNA is amber suppression tRNA, described
Selecting codon is amber codon, and described translation system also comprises the described orthogonal aminoacyl-tRNA synzyme of coding
Nucleotide sequence.
4. a host cell, it comprises the nucleotide sequence of the 1st described orthogonal aminoacyl-tRNA synzyme of coding
With corresponding orthogonal tRNA sequence.
5. the host cell as described in the 4th, wherein said host cell is that eubacteria cell, preferably escherichia coli are thin
Born of the same parents.
6. one kind produces at least one selected location fixed point specificity insertion 3, the mutain of 5-bis-fluorotyrosine
The method of matter, described method comprises the steps:
A () provides the 2nd described 3,5-bis-fluoro tyrosine translation system, and this system comprises:
(i) 3,5-bis-fluorotyrosine；
(ii) the 1st described orthogonal aminoacyl-tRNA synzyme；
(iii) orthogonal tRNA, it comprises the polynucleotide sequence shown in SEQ ID NO:1；Wherein said orthogonal aminoacyl-
TRNA synzyme with described 3, orthogonal tRNA described in the preferential aminoacylation of 5-bis-fluorotyrosine；With
(iv) encoding the nucleic acid of described target protein, wherein said nucleic acid comprises described orthogonal tRNA in selected position
At least one of specific recognition selects codon；With
(b) by described orthogonal tRNA sequence and encode described orthogonal aminoacyl-tRNA synzyme nucleotide sequence and
The nucleotide sequence encoding described target protein clones and is transformed in suitable host cell, adds 3 in the medium, 5-bis-
Fluorotyrosine, during the translation of described target protein, the orthogonal tRNA of 3,5-bis-fluorotyrosine aminoacylations identifies and compiles
Selection codon and 3 on the mRNA of the described target protein of code, 5-bis-fluorotyrosine, thus mediate 3,5-bis-fluoro cheese
Propylhomoserin fixed point specificity inserts the amino acid position that described selection codon is corresponding, thus produces in selected location containing 3,5-difluoro
Described target protein for tyrosine.
7. the method as described in the 6th, wherein said orthogonal tRNA is amber suppression tRNA, and described selection password
Son is amber codon.
8. producing containing 3, the method for the protokaryon protein tyrosine kinase mutant of 5-bis-fluorotyrosine, it utilizes the 6th
Method described in Xiang, the nucleotide sequence of coding protokaryon protein tyrosine kinase mutant wherein used comprises in selected position
The selection codon of described orthogonal tRNA specific recognition, during kinase whose translation, 3,5-bis-fluorotyrosine fixed points are inserted
To the amino acid position that described selection codon is corresponding, thus produce and contain 3 at select location, the protokaryon of 5-bis-fluorotyrosine
Protein tyrosine kinase mutant.
9. by the 8th described method obtain containing 3, the protokaryon protein tyrosine kinase sudden change of 5-bis-fluorotyrosine
Body, its aminoacid sequence is SEQ ID NO:8.
10. the method identifying the Tyr phosphorylation site of target protein, described method includes the side utilizing the 6th
Presumption in described target protein is carried out the tyrosine of phosphorylation and is substituted by 3 by method, and 5-bis-fluorotyrosine thus obtains containing 3,
The target protein mutant of 5-bis-fluorotyrosine, utilizes this target protein mutant as tetraalkylammonium fluoride, passes through19F-NMR
Technology detects 3, the phosphorylation of 5-bis-fluorotyrosine, thus identifies whether described tyrosine site is tyrosine phosphorylation
Site.
The nucleotide sequence of the orthogonal aminoacyl-tRNA synzyme of 11. codings the 1st.
13. nucleotide sequences as described in the 11st, it is SEQ ID NO:3.
3, beneficial effect
This research has obtained a kind of orthogonal aminoacyl-tRNA synzyme by screening, and, have developed 3 on this basis,
5-bis-fluoro tyrosine translation system.Can at target protein such as by this system, protokaryon protein tyrosine kinase Etk
Middle fixed point specificity insertion 3,5-bis-fluorotyrosine, produce 574 and contain 3, the tyrosine kinase of 5-bis-fluorotyrosine is dashed forward
Variant, as tetraalkylammonium fluoride, such that it is able to pass through19F-NMR technology detects and quantifies tyrosine phosphorylation level.
The new method of our this detection tyrosine phosphorylation level has the advantage of many compared with traditional method.First,
Can directly detect and the tyrosine phosphorylation level of a certain specific site of quantified goal protein.Secondly, sample can be at any time
Frozen, carry out the most again19FNMR spectrum analysis, as such, it is possible to shorten the preparation time of sample to greatest extent, in order to more accurate
The phosphorylation level of ground Quantitative Western quality sample.3rd, due to19The high sensitivity of F, relative to the chemical shift of environment respectively to different
Property and19F-19The direct-coupled shortage of F, unit point is integrated19F combines NMR analysis can be more effectively for illustrating albumen
Dynamic characteristic.Finally, NMR research potentially contributes to disclose eucaryon and protokaryon protein tyrosine kinase activity state and non-live character
Dynamic exchange between the kinetic characteristic of state, and these states.The advantage of these uniquenesses enables us to obtain positive evidence
Prove and quantify protokaryon protein tyrosine kinase Etk active center phosphorylation level, and this former cannot realize.
Further, the method for this monitoring tyrosine kinase activation and activity is highly sensitive, and selectivity is strong, can be tyrosine kinase/end
The new inhibitor screening of thing protein-interacting provides effective approach.
Accompanying drawing explanation
From detailed description below in conjunction with the accompanying drawings, features described above and the advantage of the present invention will be apparent from, wherein:
Fig. 1 is orthogonal the 3 of the present invention, the crystal structure of 5-bis-fluorotyrosine aminoacyl-tRNA synthetase (F2YRS)
Figure；
Fig. 2: Fig. 2 A is the acid urea-polyacrylamide gel electrophoresis figure of external aminoacylation effect, and Fig. 2 B is that F2Y-is green
The SDS-PAGE of fluorescin, Fig. 2 C and Fig. 2 D are the mass spectruies of F2Y-green fluorescent protein；
Fig. 3 is 3,5-bis-fluorotyrosine (F2Y) (figure below) and o-phosphoric acid-3,5-bis-fluorotyrosine (pF2Y) (upper figure)
's19F-NMR spectrogram；
Fig. 4: Fig. 4 A is wild type protokaryon protein tyrosine kinase (Etk, swimming lane 1) and Etk-574-F2Y (swimming lane 2)
SDS-PAGE, Fig. 4 B is that (upper figure is pTyr for the Western detection figure of Etk-574-F2Y and tyrosine phosphatase PTP1B
Antibody, figure below is His antibody), Fig. 4 C is Etk-574-F2Y phosphorylation19F-NMR spectrogram, Fig. 4 D is Etk-574-F2Y dephosphorization
Acidifying19F-NMR spectrogram；
Fig. 5 is orthogonal tRNA, wild type tyrosyl-t RNA synthetase, the orthogonal aminoacyl-tRNA synzyme of the present invention, green
Color fluorescin mutant, protokaryon protein tyrosine kinase mutant and the sequence of Protein-tyrosine-phosphatase.
Detailed description of the invention
It is further elucidated with the present invention by the following examples.However, it should be understood that described embodiment is merely illustrative
Purpose, be 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, chemical reagent used in following embodiment is can
By the reagent of the analytical pure rank that commercial sources is buied.
Embodiment 1:o-phosphoric acid-3, the chemosynthesis of 5-bis-fluorotyrosine (pF2Y)
Take 100mM phosphorus oxychloride, 100mM3,5-bis-fluorotyrosine (purchased from gill biochemical corp, Shanghai) and 500mM hydrogen
Sodium oxide, is dissolved to 5mL, and 1h is then stirred at room temperature.Collect aqueous phase, by the isolated and purified white powder that obtains of HPLC, productivity
10%.(YMC AA12S052503WT column, 12ml/min flow rate, from10%to90%CH3CN, 0.1%
TFA (w/v) in water, over the course of 30 min) .MS:m/z:298 [M+H]+；1H-NMR (600MHz,
D2O): 7.03 (d, 2H) 4.01 (dd, 1H) 3.21 (m, 2H).
Needed for above synthetic reaction, chemical reagent is if no special instructions, is purchased from Beijing Chemical Plant, is more than analytical pure
Rank.
Embodiment 2: evolution F2Y specificity aminoacyl-tRNA synthetase
In order in gene locus specificity insert F2Y, need in E.coli host cell used introduce aminoacyl-
TRNA synzyme/tRNA is orthogonal right, and this is orthogonal to deriving from Methanococcus jannaschii (Methanococcusjannaschii) amber
Amber suppression tyrosyl tRNA (MjtRNACUA Y(its aminoacid sequence is SEQ to)/tyrosyl-t RNA synthetase for MjYRS, wild type
ID NO:2) right.MjYRS mutation library builds and receives chloramphenicol resistance pBK plasmid (purchased from U.S. scripps institute Peter at card
G.Schultz laboratory) in, on this plasmid between promoter and the terminator of E.coli glutamine synthetase.Made
Synzyme mutation library be pBk-lib-jw1 storehouse, the construction method of this mutation library is: select 6 sites on MjYRS gene
(Y32, Leu65, Phe108, Gln109, Asp158, and Leu162) introduces NNK sudden change (N=A+T+C+G；K=T+G), other 6
Individual site (Ile63, Ala67, His70, Y114, Ile159, Vall64) or random mutation are Gly or keep constant and (see
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).
The aminoacyl-tRNA synthetase of specific recognition F2Y of being evolved by positive-negative selection.Just screening plasmid to comprise
MjtRNACUA Y, the chloramphenicol acetyl transferasegene of TAG sudden change, start the T7RNA of the amber mutation of expressing green fluorescent protein
Polymerase, tetracycline resistance gene.Negative screening plasmid comprises MjtRNACUA Y, amber mutation bud under arabinose operon
Spore bacillus RNase gene, and ampicillin resistance gene.Carry out 3 and take turns positive-negative selection: include and just screen plasmid
E.coli DH10B cell is as just screening host cell.Cell electricity turns pbk-lib-jw1 storehouse, SOC culture medium (2% (W/V) pancreas
Peptone, 0.5% (W/V) yeast powder, 0.05% (W/V) NaCl, 2.5mM KCl, 10mM MgCl2, 20mM glucose) and 37
DEG C cultivate 1 hour.Use the minimal medium (formula of GMML minimal medium: M9 salt/glycerol: 764gNa afterwards instead2HPO4.7H2O
Or 30g Na2HPO4, 15g KH2PO4, 2.5g NaCl, 5g NH4Cl, 50ml glycerol, autoclaving, pH7.0；1M MgSO4:
Autoclaving；50mM CaCl2: autoclaving；25mM FeCl2: filtration sterilization；0.3M leucine: be dissolved in 0.3M NaOH,
Filtration sterilization；1L liquid GMML culture medium: 200ml M9 salt/glycerol, 2ml MgSO4, 2ml CaCl2, 2ml FeCl2, 1ml is bright
Propylhomoserin) wash twice, bed board solid minimal medium (in liquid GMML culture medium add 500ml3% agar powder, 1mM F2Y,
50mg/L kanamycin, 60mg/L chloromycetin, 15mg/L tetracycline), cultivate 60 hours for 37 DEG C.Collect cell, extract plasmid
DNA, electrophoretic separation, glue reclaims.Then, the pBK-lib-jw1 through just screening is transformed into comprises negative screening plasmid
In DH10B competent cell.SOC culture medium is recovered 1 hour.Coated plate comprises 0.2% arabinose (purchased from sigma public affairs afterwards
Department) LB solid medium (every liter of culture medium tryptone Han 10g, 5g yeast powder, 10g NaCl).37 DEG C of cultivation 8-12 are little
Time.Repeat 3 altogether to take turns.
Last is taken turns just to screen and chooses 384 clones, and some plate is containing 1mM F2Y, chloromycetin 60,80 respectively, 120,160 μ
On the GMML solid medium of g/mL, and do not comprise F2Y but comprise chloromycetin 0, the GMML solid culture of 20,40,60 μ g/mL
Base.Select and grow in the culture medium at 1mM F2Y160 μ g/mL chloromycetin, and at 0mM F2Y, concentration is more than 20 μ g/mL chlorine
The clone not grown in mycin culture medium verifies further.Finally choosing 1 clone, insert 3,5-bis-fluorotyrosine is imitated
Rate is the highest, and order-checking shows, the aminoacid sequence of the aminoacyl-tRNA synthetase mutant (F2YRS) that clone is comprised is SEQ
Shown in ID NO:4, wherein mutational site is Tyr32Arg, Leu65Tyr, His70Gly, Phe108Asn, Gln109Cys,
Asp158Asn and Leu162Ser.
It should be appreciated by those skilled in the art that in the present invention, except the aminoacid sequence shown in SEQ ID NO:4 it
Outward, term " orthogonal aminoacyl-tRNA synzyme " or " orthogonal 3,5-bis-fluorotyrosine aminoacyl-tRNA synthetase " also include
The examples of conservative variations of aminoacid sequence shown in SEQ ID NO:4, if described examples of conservative variations have with shown in SEQ ID NO:4
The identical enzymatic activity of aminoacid sequence；And also include the aminoacid sequence shown in SEQ ID NO:4 through one
Or multiple amino acid whose replacement, lack or add and there is the enzymatic activity identical with the aminoacid sequence shown in SEQ ID NO:4
The aminoacid sequence derivative by the aminoacid sequence shown in SEQ ID NO:4.
Meanwhile, the present inventor have also resolved the crystal structure (Fig. 1) of F2YRS, provides for F2YRS specific integration F2Y
Architecture basics.
Embodiment 3: external aminoacylates function analysis
In order to verify that F2YRS integrates high efficiency and the fidelity of F2Y in target protein, We conducted external aminoacyl
Change function analysis.Take 50mM sodium chloride, 20mM magnesium chloride, 4mM dithiothreitol, DTT, 2mM ATP, 10 μMs of Tyr tRNA, 3 μMs
F2YRS and 2mM tyrosine or F2Y, be dissolved in the Tris buffer of 20mM, pH8.0, hatches 1h for 37 DEG C.Reactant liquor is carried out
24h acidity carbamide Polyacrylamide Gel Electrophoresis, as shown in Figure 2 A, F2YRS can only integrate F2Y to result, and cannot integrate
Tyrosine.
Embodiment 4: express F2Y-green fluorescent protein and Mass Spectrometric Identification
By orthogonal tRNA (SEQ ID NO:1) and the nucleotide sequence (SEQ ID NO:3) of coding F2YRS that screens
It is building up on pEVOL carrier (purchased from U.S. scripps institute Peter G.Schultz laboratory), encoding green fluorescent egg
White nucleotide sequence (151TAG) (SEQ ID NO:5) is building up to pET carrier (purchased from U.S. scripps institute Peter
G.Schultz laboratory) on, then cotransformation is in DH10B cell (purchased from Quan Shi King Company).Picking is single is cloned in 37 DEG C
Cultivate OD600When approximating 1.2, in LB culture medium, add 1mM IPTG, 0.02% arabinose (purchased from sigma company)
And 0.5mM F2Y cultivates cell, comparison is added without F2Y.After 8 hours, receive bacterium, Ni-NTA purifying protein, and use SDS-PAGE
Electrophoretic analysis (Fig. 2 B).
It was found that only just can be purified into the green fluorescent protein of total length in the culture medium exist F2Y, this illustrates sieve
The F2YRS elected can specific identification F2Y.In LB culture medium, the productivity of F2Y-green fluorescent protein is 60mg/L,
And the productivity of wild type Green Fluorescent albumen is 100mg/L.It is merely inserted into 151 of green fluorescent protein to detect F2Y
Amber mutation site, we have carried out ESI-TOF Mass Spectrometer Method to 151-F2Y-green protein, and testing result molecular weight is
27746Da (Fig. 2 C, D), coincide with the molecular weight 27746Da calculated.
Embodiment 5: express F2Y-protokaryon protein tyrosine kinase mutant and tyrosine phosphorylation detection
We choose protokaryon protein tyrosine kinase Etk C-terminal cytoplasmic domain fragment (451-726, nucleotide sequence
As shown in SEQ ID NO:7), construct Etk mutant by gene engineering method, wherein 574 sport TAG termination codon
Son, then inserts F2Y by the same procedure in embodiment 4 at 574 fixed point specificitys of Etk mutant, expresses and produce sudden change
Albumen Etk-574-F2Y (aminoacid sequence is as shown in SEQ ID NO:8), productivity is 12.5mg/L, and the productivity of wild type Etk
For 20mg/L (Fig. 4 A).
In order to verify that can 574 tyrosine in Etk active center carry out autophosphorylation, we take mutain Etk-
574-F2Y, incubates for 25 DEG C in the buffer (containing: 20mM Tris, 20mM NaCl, 2mM ATP and 5mM MgCl2) of pH8.5
Educate 30min.As comparison, we add 0.125mg/mL Protein-tyrosine-phosphatase in mutain Etk-574-F2Y
PTP1B (obtained by conventional molecular biological method clonal expression, or can also be commercially available, aminoacid sequence is such as
Shown in SEQ ID NO:9) carry out dephosphorylation, then the buffer in pH8.5 (contains: 20mMTris, 20mM NaCl and 1mM
EDTA) 30min is hatched for 25 DEG C in.After hatching end, taking a small amount of mixed liquor respectively and carry out Western checking, remaining sample is at once
Carry out after lyophilizing19F-NMR analyzes.Meanwhile, we pF2Y newly synthesized in taking F2Y and embodiment 1 is carried out19F-NMR analyzes, and makees
Comparison for checking F2Y whether phosphorylation.Result such as Fig. 3 and Fig. 4 C, shown in D, F2Y's19F chemical shift is-133.1ppm, and
PF2Y is-126.7ppm (Fig. 3).Fig. 4 C and D shows, contain only the mixed liquor of Etk-574-F2Y respectively-122.3ppm and-
All occur in that signal peak at 134.5ppm, and add the mixed liquor of PTP1B, signal peak, explanation only occur at-134.5ppm place
574 two fluorotyrosines of Etk-574-F2Y there occurs autophosphorylation in buffer, this conclusion and the inspection of western
Survey result completely the same (Fig. 4 B)., being found by signal intensity relative analysis, only two fluorotyrosines of 3.8% are sent out meanwhile
Given birth to phosphorylation, this be previously reported by the phosphorylation level at alleged protokaryon protein tyrosine kinase activity center is the lowest is also
Consistent.
Although it should be understood that with reference to its exemplary embodiment, the present invention carried out particularly shown and described,
It should be understood by those skilled in the art that without departing substantially from by the spirit of the present invention as defined in the claims and model
Under conditions of enclosing, the change of various forms and details can be carried out wherein, the combination in any of various embodiment can be carried out.
Claims (10)
1. an orthogonal aminoacyl-tRNA synzyme, its aminoacid sequence is as shown in SEQ ID NO:4.
2. one kind 3,5-bis-fluoro tyrosine translation system, described system comprises:
(i) 3,5-bis-fluorotyrosine；
(ii) orthogonal aminoacyl-tRNA synzyme described in claim 1；
(iii) orthogonal tRNA, it is as shown in SEQ ID NO:1；Wherein said orthogonal aminoacyl-tRNA synzyme is with described 3,5-
Orthogonal tRNA described in the two preferential aminoacylations of fluorotyrosine；With
(iv) nucleic acid of encoding target protein, wherein said nucleic acid contains at least one of described orthogonal tRNA specific recognition
Select codon.
3. translation system as claimed in claim 2, it is characterised in that described selection codon is amber codon, and institute
State translation system and also comprise the nucleotide sequence encoding described orthogonal aminoacyl-tRNA synzyme.
4. a host cell, it comprises the nucleotide sequence of coding orthogonal aminoacyl-tRNA synzyme described in claim 1
With corresponding orthogonal tRNA sequence.
5. host cell as claimed in claim 4, wherein said host cell is eubacteria cell.
6. host cell as claimed in claim 5, it is Bacillus coli cells.
7. one kind produces at least one selected location fixed point specificity insertion 3, the mutein of 5-bis-fluorotyrosine
Method, described method comprises the steps:
A () provides 3 described in claim 2,5-bis-fluoro tyrosine translation system, this system comprises:
(i) 3,5-bis-fluorotyrosine；
(ii) orthogonal aminoacyl-tRNA synzyme described in claim 1；
(iii) orthogonal tRNA, it is for shown in SEQ ID NO:1；Wherein said orthogonal aminoacyl-tRNA synzyme is with described 3,5-
Orthogonal tRNA described in the two preferential aminoacylations of fluorotyrosine；With
(iv) encoding the nucleic acid of described target protein, it is special that wherein said nucleic acid comprises described orthogonal tRNA in selected position
Property identification at least one select codon；With
B () is by described orthogonal tRNA sequence and the nucleotide sequence and the coding that encode described orthogonal aminoacyl-tRNA synzyme
The nucleotide sequence of described target protein clones and is transformed in suitable host cell, adds 3 in the medium, 5-bis-fluoro
Tyrosine, during the translation of described target protein, the orthogonal tRNA recognition coding institute of 3,5-bis-fluorotyrosine aminoacylations
State the selection codon and 3 on the mRNA of target protein, 5-bis-fluorotyrosine, thus mediate 3,5-bis-fluorotyrosine
Fixed point specificity inserts the amino acid position that described selection codon is corresponding, produces in selected location containing 3,5-bis-fluorotyrosine
Described target protein.
8. method as claimed in claim 7, wherein said selection codon is amber codon.
9. producing containing 3, the method for the protokaryon protein tyrosine kinase mutant of 5-bis-fluorotyrosine, it utilizes claim
Method described in 7, the nucleotide sequence of coding protokaryon protein tyrosine kinase mutant wherein used comprises in selected position
The selection codon of described orthogonal tRNA specific recognition, during kinase whose translation, 3,5-bis-fluorotyrosine fixed points are inserted
To the amino acid position that described selection codon is corresponding, thus produce and contain 3 at select location, the protokaryon of 5-bis-fluorotyrosine
Protein tyrosine kinase mutant.
10. the method identifying the Tyr phosphorylation site of target protein, described method includes the side utilizing claim 7
Presumption in described target protein is carried out the tyrosine of phosphorylation and is substituted by 3 by method, and 5-bis-fluorotyrosine thus obtains containing 3,
The target protein mutant of 5-bis-fluorotyrosine, utilizes this target protein mutant as tetraalkylammonium fluoride, passes through19F-NMR
Technology detects 3, the phosphorylation of 5-bis-fluorotyrosine, thus identifies whether described tyrosine site is tyrosine phosphorylation position
Point.
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