JPH04504651A - A method for site-specifically introducing non-naturally occurring amino acids into proteins - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] A method for site-specifically introducing amino acids that do not exist in nature into proteins. U.S. Patent Application No. 27, filed November 18, 1988. ．． 455 and U.S. This is a continuation-in-part of patent application number 273.786. These related applications are reference text is incorporated herein as a reference.

This invention was created by the Office of Naval Research (Office of Naval Research). ONR granted by l　Re5earch):+Mettract N00014- 86-0522 and DOE grant No. DE AC03- This was done with government support based on 76SFOO09g. The government is this Have a fixed right to the invention.

Oihi Tate Satsuri Tate Mutual This invention relates generally to the biochemistry of proteins, and more specifically to the specificity of enzymes. modifying the structure of naturally occurring proteins to modulate their properties and activities; This invention relates to a method for effectively site-specifically modifying proteins.

Background of the invention Classically, biochemists modify protein function by chemically modifying purified proteins. This has been done by changing the molecule or by selecting naturally occurring mutants.

Chemical modifications typically involve similar modifications to highly reactive and solvent-soluble amino acid side chains. It will be done. However, often the desired modification site is not available or Certain modifications are chemically impossible. The low specificity of the modification reaction is an advantage of this method. dramatically hinders effectiveness. Furthermore, naturally occurring variants are rare and usually Naturally occurring amino acid residues require thorough analysis to determine their different characteristics. The conversion of is usually limited.

With the advent of molecular biological engineering, specific amino acids have been can be incorporated into proteins and peptides during the elongation process of the peptide chain. Other possible methods have been improved. Peptide synthesis methods and semi-synthesis methods are new have been used to introduce amino acids into very small proteins and peptides. Ami Modification using an analog with the function of noacyl transfer RNA (tRNA) These amino acids have been uniformly incorporated into peptides and proteins. Further attached In addition, some unnatural amino acids can lead to chemically incorrectly acylated tR Incorporated into the dipeptide using NA.

All of these methods suffer from one or more of the drawbacks described below. Ta. That is, lack of site specificity when introducing a new amino acid, and unevenness of modification sites. sufficient analysis to accurately determine the specificity, low modification efficiency, and the site and nature of the substitution. the need to Possibilities for modification are very limited. Thus, tan, fri There is a need for improved methods for making specific modifications at desired sites of quality. child The invention described herein satisfies these needs, and may also satisfy other needs. It is.

Summary of the invention The invention described here allows us to transform unnatural amino acid analogs into tanno-rich parts. A new method for specific uptake has been provided. The method consists of the following steps: .

(a) Convert the preselected codon into a tannoic protein - introduced into at least one site on the RNA sequence, and (b) unnatural amino acid analogs at preselected co-locations of the polypeptide chain being synthesized. Tan containing an aminoacyl-tRNA analog that can be polymerized at the site of Don. Translating this metsenger RNA sequence using a protein synthesis system.

The protein synthesis system mainly uses an in vitro tanno-crystalline synthesis system. An example of a f-codon is a stop codon, such as UAG (Annoy), in advance. Insert in the determined part.

Typically modified natural amino acids as non-naturally occurring amino acid analogs modified amino acids, those modified with uncharged amino acids, and those modified with acidic amino acids. , modified basic amino acids, non-alpha amino acids, φ ℃ 1 is φ angle Amino acids converted into amino acids, amino acids containing the following functional groups, i.e. nitro, amidi from quinones, hydroxylamines, quinones, aliphatic compounds, cyclic and unsaturated compounds. The chosen ones are chosen. Under preferred conditions, aminoacyl-tRNA analogs The protein synthesis system recognizes unique, preselected codons. is an aminoacyl-tRNA molecule that can also contain preselected codons. Foods typically contain tannoic substances with a molecular weight of 10,000 Daltons or more. Rume1. Senji + - is introduced at one location in the RNA. Doesn't exist in nature (Asahi amino acid analogs 1, substrate binding sites, enzyme active sites, proteins and proteins binding site, cofactor binding site or ligand (agonist or antagonist) (LSIt antagonist with agonist) Can be located within Gström.

Another aspect of the invention is that the preselected one (or more) The stoichiometrically uniform substitution of one amino acid analog that does not exist in nature Contains new proteins (usually larger than 10 kilodaltons) that have been acquired. Tannokuri The analysis of the physical or biochemical properties of The static properties of the tide chain, the mechanism of enzymatic reactions, and the ligand binding of tide chains. Specificity, interaction of amino acid residues of target protein with substrate, Tan7+ Holding (folding/formation of higher-order structure) of lithium, Tan 1 < lithium and others It is possible to determine properties such as tannin content and interaction with nucleic acids and sugars. Noriyoshi Typically, the natural (approximately 100 oz It can be analyzed within the range of Trom.

Another aspect is that, in the steps described below, this invention We developed a method for alternatively replacing selected amino acid sites with various other amino acids. provide. : a) Select a site in Metzenger RNA that corresponds to a preselected amino acid site. , create metsenger RNA that replaces the mistranslated codon. and , b) Metsenger 1 iNA must contain aminoacyl-tRNA analog 2 It is translated into protein using a translation system that is more than 1 c + LIt. Here, one The tanno<ri quality created by the translation system of Compared to the quality, the preselected amino acid sites are different.

Under desirable conditions, one amino acid site is replaced and a different translation system (or The difference between each protein made by Determined in advance by selecting a non-naturally occurring amino acid analog that is linked to be done.

Substitutions of non-naturally occurring amino acids include, for example, D-phenylalanine, (S)- p-nitrophenylalanine, (S)-homophenylalanine, (S)-p -Fluorophenylalanine, (S)-3-amino-2-benzylpropion An acid (X is (S)-2-hydroxy-3-phenylbrobionic acid, etc.).

Furthermore, another feature of this invention is to create aminoacyl-tRNA analog molecules. It is related to the way in which The method consists of the following steps.

a) 2° of the 3° terminal nucleotide of a multinucleotide molecule (MNM) or preselected by aminoacylation linkage to the 3-ribonyl hydroxyl site. b) combining amino acid analogs that do not occur naturally; and b) amino acid analogs. The end of the converted multinucleotide molecule (aminoacyl-MNM) is excised. tRNA molecules (tRNA (-Z)). In this way it has a function An aminoacyl-tRNA analog molecule is synthesized.

Preferably, the multinucleotide molecule (MNM) is, for example, 5°pCpA3°, is a dinucleotide corresponding to the 3' end of tRNA. multinucleotide molecule (MNM) to a tRNA(-Z) molecule is typically a complete tRN Create A molecule. Then, tRNA (-Z) is activated after reading (run-off). ) can also be made from transcripts.

2 or 3” chain of the 3° terminal nucleotide of a multinucleotide molecule (MNM) Preselected non-naturally occurring amino acid analogs at the amino acid position Aminoacylation of the molecule consists of the following reaction steps.

a) Protecting the reactive group on MNM with a protecting agent, b) Amino acid analog protecting the non-aminoacylated reactive groups on the group with an inhibitor; C) Acylation of MNM using an amino acid analog protected by a blocking agent to do, and d) Removing the protecting or blocking agent from the protected reactive group.

Some or all of the above procedures for protecting reactive groups may be performed using the following blocking agents. or a procedure using a protective agent. That is, 0-nitrophenylsulfenyl (NSP); β-7anoethyl (EtCNO); benzyloxycarbonyl (CBZ); 9-fluoroenylameneluoki7carbonyl<FMOC) ; 2-(4-biphenyl)isopropyloxycarbonyl (BPOC), Vinyloxycarbonyl (VOC); Tetrahydropyranyl (THP); ibis/tetrahydropyranyl; and photosensitive groups such as 4-methoxy- Contains 2-nitrobenzyloxycarbamate (1ffOc). Protection processing is 0 - Use of nitrophenylsphenyl (NFS) as both a blocking agent and a protective agent. There may be cases where Furthermore, aminoacyl-MNM was bound to tRNA(-Z). The reaction may be performed using T4 RNA ligase enzyme.

Another aspect of this invention is that the aminouncle tRNA analog has the following formula: Consists of having. The formula is X-A-Y-M.

Here, X = 5' nucleotide sequence of the tRN^ molecule; A = anticodon nucleotide sequence; Otido; Y = 3' nucleotide sequence of the tRNA molecule, e.g. 5-pcpcpA-3' ; M: Amino acid analogs selected from the following groups 1) Modified uncharged natural amino acids amino acids 11) Modified acidic natural amino acids ii+) Non-alpha amino acids These analogs are incorporated into the polypeptide chain during synthesis as M components, Subsequently (can act as an acceptor for peptide polymerization, e.g., (S)-p -tRNAPc’u” aminoacylated by nitrophenylalanine The analog corresponding to lI is synthesized as follows.

a) X is 5 of tRNAPchu- containing the D loop and anticodon loop part Consisting of parts; b) A (anticodon) is from the trinucleotide 5'-pcpUpA-3° c) Y is part of the anticodon loop, variable loop, TφC loop and It consists of 3 parts of tRNA'c'u・^ containing the acceptor stem; d) M is (S)-p-nitrophenylalanine.

The invention further provides a translation system containing such an aminoacyl-tRNA analog. include. It also includes a simultaneous transcription and translation system. In this system, what is synthesized in the transcription system is 0 drawings translated with a translation system containing such an aminoful tRNA analog. easy explanation Figure 1 shows a method for site-specifically introducing amino acids that do not exist naturally into proteins. It is a schematic diagram.

FIG. 2 is a diagram of the synthesis of aminoalized pA.

Figure 3 shows the vector used for in vitro expression of β-lactamase, plasmid p. It is a figure which shows the creation method of SG7. The a segment is pKKK223-3 (broth-ri said Hosie.

(1984) Proc, Natl, Acad, Sci, USA, I 25 containing the tacpromoter (ll:6929) 9-base pair (bp) BamHI-EcoR1 portion. b segment is RTE Mβ-lactamase (Sutcliff, (197g) Proc, Nat l, Acad, Set, USA, 15:3131: Ambler and 5c ott, (1978) Proc, Natl, Acad, Sci, t lsA, IS:313Z; and Politt and Zalkin, (19 83) J, Bacteriol, 153:27) including the first part of 37 6 base bare (bl)) Ssp1 part (pTG2dell (Kadonaga) et al, (1984) J, Biol, Chet 2T9: 2149), which is linked to the EcoRI-Pvul fragment of -There is a deletion of 63 bases corresponding to 21 amino acids in Darcy fence. C segment is pT7-3 (Tabor and Richardson, (1985) P roc, Natl, Acad, Set, LISA, 82:LO74) β-ratatama [ze 1386 base pairs of Pvul-H containing the remainder of the gene and the Co1E1 replication origin. aell fragment. The d segment is pGPI-2 (Tabor and Rie Hardson, (1985) Proc, Natl, AeadSet, USA, 82:1074) Tn903 (Oka at al, (198 1) J, Mo1. Biol, 147+21V)? 1laal 1-)1 of 1430#A group containing the kanamycin resistance gene of et al. Contains the ae17 fragment. This gene is under the transcriptional control of the tack promoter. It looks like it has not been placed. The C segment is a 289 base pair Ha from pT73. e　　! -Pvu I +7 Ragment (Ba ■ To create only the old site a conjugated to the plant-end BamH1 portion of the segment).

The mutant strain of PheJ6 (* in the figure) is Eckstein. Law (Nakamaye and Eckstein, (19116) Nucl, Ac1ds, Res, 14:9679). Phe66 A synthetic EcoRI-Hinel+7 fragment of 204 base pairs containing the codon M1 It was introduced into 3mp1g. Three oligodeoxynucleotides 5-ATCA TTGGATAACGTTCTT-3'.

5°-ATCATTGGA ACGTTCTT-3', 5°-ATCATT GG Rainbow AACGTTCTT-3' (underlined bases are different from the base sequence of the wild strain) ) produced F66Y, F66A, and Ff+6u mutant strains, respectively. It was used to The mutagenesis efficiency was 100% for Tyr66 and 100% for Ala6. It was 83% with 6 and 60i with TAG66.

Figure 4 shows a method for in vitro synthesis and purification of truncated β-lactamase.

1st row: Crude in vitro synthesized reaction product; 2nd row: β-lactamase purified in vitro Product; 3rd row: β-lactamase synthesized in vivo (JMIOI/psG7) product.

Figure 5 shows the tRNA T'llE/CLIA(-CA) sequence determined using the enzyme. Indicates a column. tRNA'chu'A (-cA) uses C5'-32pl-pCp. The 3° end was labeled using the nucleotide sequence-enzyme method (A method (Donis-Kel ler, (1980) Nucl, Ac1ds, Res, 8:3H3) It was decided. The degradation products were loaded onto a 10% denaturing polyacrylamide gel.

The autoradiogram of base sequencing is shown in the figure. : 1 row without enzyme), -OH decomposition (rows 2 and 7), RNase T1 degradation (G-specific, row 3>, R Naseυ2 (A-specific, 4 rows>, RNasePhy) 4 (specific to υ+8, 5 column), RNaseB eereus (Ll+ClI, column 6). Ru. The base sequences of the anticodon stem and loop are as shown in the figure. Parts are shown in capital letters and are 0. tRNAPchu・Q(-CA) is suitable for electrophoresis Therefore, there is a nucleo that can be purified and used in chemical misacylation reactions. Chidyltransferase (Cudney and Deutscher, (19 86) J, Biol.

Chem, 261:6450) and purified by electrophoresis. Yeast PRS was used in the misacylation reaction.

Figure 6 shows the acylated and unacylated ℃1 Sable-7ser tRNA. In vitro testing is shown. The reaction (30 μL) was chilled at 0 °C as shown in Figure 4. It was held a long time ago. 3 μL of each supernatant was denatured and diluted with 12.5% SDS. Polyacrylamide gel aemmlt. (197(1) Nature (Lo After filling, the gel was dried and autoradiated. I did an ophthalmology. Row 1: pSG7 (truncated β-lactamase ); 2 rows: using pF66am without adding suppressor Reaction initiated with g) Reaction started with no added material. 1.2 and 3 columns are the final evening effect concentration 19 Added 0 Ci/mol ['H]-Phe (Amersham); row 4: pF66 group and [”H]Jha (specifi l quaactivity 9.4Ci 5 μg of protein acylated using the enzyme in the presence of Phe-tRNA) For pressers (started with X.

Enzyme-based misacylation reactions (total 11,300 μL) included: 4 μMt”NApC”LI@A (30Mg, purified using gel, desalted and frozen) dried).

80MM Phenylalanine, 4 (ld Tris-HCI (p) [8.5 ), H+aM iAgclz, 45Mg/mL BSA.

3, 3+aM DTT. 2mM ATP and 22 units yeast PRS (here So, 1 unit of activity means 100 pmol of ferrite at 37°C for 2 minutes under the following conditions. Conditions for incorporating nilalanine (Phe): 2 μM tRNA'' (B oeringer Mannheim), 2mM ATP, 3. 3mM DDT, 8.　μM　ohe. 40MM Na HEPES (pH 7.4), 15mM MgC12. 25mM KCI and 50 Mg/mL BSAo (the reaction solution was heated at 37 °C for 3 minutes, then 2. Add 5M NaOAe (pH 4.5) to a final concentration of 10% v/v and incubate. response was stopped. After stopping, the reaction solution was mixed with phenol (0.25M NaOAc, pif 4. Equilibrated with 5)), Phenol: C11C13 (1・1) , immediately extracted with CHCL3, and then precipitated with Etoll.

The extraction and precipitation were repeated two more times. The tRNA is then produced using Pharmacia's It was desalted using a Studi Salting column and lyophilized. Acylated and acylated Lyophilized unsylated tRNA can be used in in vitro protein synthesis reactions. It was stored at -80°C until used.

β-lactamase activity in the reaction supernatant was measured by nitrocephin hydrolysis assay. (0°Callaghan et al., (1972) Anti +microb. Ag. Chemother. , 1:Q83.　1 unit The nitrocefuin hydrolysis activity of (1 μmol nitrocefuin hydrolyzate/ in/mL.

Bra. This corresponds to 0.61 Mg of enzyme as determined by the 7-domain assay. ).

Wardza L Beniwa 1 pSGl 44.6 2pF66amQ 3 pF66am. Non-acylation suppressor 04 pF66at acylation sub Les Asables 6.7 Figure 7 shows chemical aminoacylation of dinucleotide pCpA We will show you how. Dinucleotide pCpA is a common liquid phase phosphotriester Synthesis method (Jones et al., (1910) Tetrahedro n. 36 +3015; Van Boom and Wreesman in -01igonucleotiр■ S3’nth6SIS”, Gait (Ed.), lRL Press, W Ashington, 1984). A fully protected molecule is 4-chlorophenyl-4-N-anisoyl-2'-0-tetrahydrobyl -5-Q- [β-anoethyloxyphosphorylco/tidylyl(3'-5 °〉-[6-N, 6-N, 2'-0,3-0-tetrabenzoylyl]ade It was nonn. Next, 0-nitrophenylsulfenyl chloride (1,b +mol) and hydrethylamine (18m+1ol) are dimethyl sulfochloride Added over 6 hours to pCpA (285 μmol) dissolved in Cyd (6 kL) It was done. The reaction was carried out in 50++M ammonium acetate, pH 5 (loOaL ) was added and stopped, the solvent was removed with vacuum suction. Reversed phase HPLC purification (HPLC conditions A = 15IIM ammonium acetate pH 5, BllMeC N, gradient end = 0-15% B for 60 min; 15-30% B for 30 min, flow Output speed = 8 L/sin, Column = Wanotman Partinl 10M-2010 1500DS-3,), 65% NFS-pCpA, 22% pCpA I got it. NPS-pCpA was desalted by reverse phase 11PLc (HPLC conditions : A=H20, BllMeCN, gradient end=O~is%B for 60 minutes; 15-30% B for 30 minutes, flow rate i + 8 ■L/■in, column = Wanoto Manpertinle 10M-20101500DS-3,), then download was passed through a column (LL+ type).

0-nitrophenylsulfenylphenylalanine (74 μmol) and N,N- Carbonyl diimidazole (83 μmol) was dissolved in anhydrous dimethyl sulfochloride under nitrogen. Mixed on the side (320 μL) for 30 minutes. Next, add NFS-pCpA to the solution. (15 μmol,) lithium salt (repeatedly evaporated to dryness in toluene) was added. It was. The reaction solution was stirred at 50°C for 8 hours under nitrogen, then 50mM ammonia was added at 0°C. Umacetate, pH 5 (2 mL) was added to stop the reaction, and after lyophilization, reverse phase HPL was performed. C (HPLC conditions: fi, 50mM ammonium acetate, B=MeC N, gradient end = 0-70% B for 70 minutes, outflow rate = ll++L/win , , Column - Whatman Partial 10M-20101500DS-3,) Therefore, it is purified and the desired product is recovered with 38% of the reaction starting material and 16% efficiency. It was done. After lyophilization, this product (2.4 μmol) was dissolved in 40 mM sodium thiol. sulfate, dissolved in 50 IIM sodium acetate, pH 4,5 (2 mL). The mixture was dissolved and stirred for 1 hour. Reverse phase HPLC (HPLC conditions: A=8mM H OAc, B = MeCN, gradient end - 0 to 30% B for 45 minutes, flow rate =4mL/■in, Column=Wasotman Bartinlu 10M91500DS- Acylated pcpA deprotected by 3,) was recovered with an efficiency of 81%.

All products were analyzed by UV, NMR and 2D NMR.

FIG. 8 shows the purification of Phe66β-lactamase synthesized according to FIG. 1. β - Lactamase was chemically acylated according to the method described in Figure 4 900 μL of pF66am-supplemented with 150 μg of Phe-tRNAcuA Purified from addition reaction. Typical efficiency is 0.3-0.7 μg (7-ts%) Purified enzyme was obtained starting from 45 μg of crude reaction.

The sample (550-200n/bind) was made of 12.5% 5DS-Polyacrylic Midgel (Laemw+li.

(1970) Nature (London), 227:680), in Therefore, after electrophoresis, silver staining was performed. Diffusion of molecular weight Mr・67000 and 60000 This band is an artifact usually observed in high-grade silver staining (Mer. ril et al, (1983) Methods Enzymol, 96:230) o Row 1: Crude reactant in test tube; Row 2: Purified reactant in test tube Synthetic β-lactamase; Row 3: Purified in vivo synthetic β-lactamase (JM IOI/pSG7).

Figure 9 shows the wild-type and inhibitory β-lactamase tributyl-degrading peptide pinepine. A diagram of the Wild-type β-lactamase is [3-old-phenylalanino in the presence of pSG7 was uniformly labeled by an in vitro protein synthesis system. Figure 4 Guelph with Sephadex G-77 (Phar-maeia) as shown in Purified using filtrane 3 and chromatofocusing chromatography. The in vitro synthesized product was spiked with unlabeled β-lactamase prior to purification. . The sequence of β-lactamase from pSG7 is shown below. The trypsin-digested part is Suggested by spaces. Peptides containing phenylalanine (Phe) are in bold It is shown in Phe66 is underlined: MSHPETLV K VK DAEDQLGARVGYIELDLNSGlf ILESFRPE ERFPMMSTFIVLLCGAVLSRVDAGQEQLGRRIHYSQ NDLVEYSPVTEK HLTDGMTVRELCSAITMSDNTA ANLLLTTIGGPK ELTAFLII) 1MGDHVTRLDRWEP ELNEAIPNDERDTTMPAAMATTLRK LLTGELLTLA SRQQLIDlfMEADK VAGPLLR5ALPAGWPIADI 5 GAfER GSRGHAALGPDGKPSRIVVIYTTGSQATMDERNRQI AEIGASLIK HW0to! The peptide after I-pusin degradation was prepared using Pharmacia P Separated by ep RPC515 column. The absorbance of the column effluent is 25 4n■ (Panel A), and fractions of 0.5 mL were collected to determine radioactivity. (Panel B). Radiolabel-inhibited β-lactamase is [”H]- Phe.

In the presence of tRNAPchu-, pF66a+* was obtained by in vitro reaction. Synthesized. Purification of inhibited β-lactamase and tributyl degradation were performed in the same way. . However, the difference is that a 6000 CPM label-inhibiting enzyme was used for the decomposition reaction ( Panel C).

FIG. 1O shows an analysis of native and mutant β-lactamases. Wild type and Ph e66-inhibited β-lactamase is pSG7 or PpF66a7Ph, respectively. It was synthesized in vitro in a reaction using e-tRNAcua and then purified. Initial The ratio of the nitrocefin hydrolysis reaction was 50 μM sodium phosphine at 24 °C. p1177 in the presence of 0.5% DMSO and substrate in the range of 25-250 μM It has been determined. The values of KM and vll, adie-11ofste plots). Also, determine the KesL value. The Bradford quantification method for enzymes was used to determine.

The reaction rate constant of the mutant enzyme was determined as follows. 15μCi [”S ] - In vitro reactions (60 μl) containing menonine (Amersham) were added to pF 66Y, pF66 carrier/Phe-tRNAPChU”A, pF66g/p-F Phe-tRNAPc''u-, pF66u/p-N02-Phe-tRNAPc 'u'n.

pF66 prefecture) IPhe-LRNAPchu”o, pF66!!/PLA-tR NA’c”u・A, pF66L7ABPA-tRNAPch P1 or pF66am/D-Phe-tRNA'chu- and incubated at 37°C. was heated for 30 minutes. The values of KII and V□8 are determined by heating reaction using crudely purified enzyme. Immediately thereafter, the decision was made as described above.

To quantify the amount of enzyme, the crude reaction solution is precipitated with trichloroacetic acid, and then the unbound enzyme is precipitated with trichloroacetic acid. Measurements were made using the first precipitate after washing at 90 °C to remove debris (Pra tt in” Transcript and Translatio n”, Hanes and Higgins (Eds,), IRk Pr ess.

Oxford, 1984. pp, 179-209), Phe66 enzyme Measurements of isotope label binding were determined using the Bradford quantitative method.

5t-880s-' and the amount of bound radioactivity per μ++ol of the enzyme (typical was used to calculate 5-Ci/μmol). This value is then This was used to quantify the mutant enzyme based on the measured bound radioactivity. shown in the figure The values shown are the average of three experiments.

Detailed description of preferred embodiments This invention is a new type of tannin that contains amino acids that do not exist in nature in specific parts. We provide a new synthesis method. This method uses a non-naturally occurring amino acid for the desired purpose. Modifications that allow the polymerization of amino acids into specific codon portions of an mRNA sequence Mainly used is ami/acyl-tRNA. Using this method, a large variety of Selectively introduce various amino acids that do not exist in nature into proteins of interest. You will be able to do so. This method uses chemicals to target specific, selected sites on a protein. A stoichiometrically sufficient and uniformly substituted protein can be provided. This person The law is simple in nature and can also be modified in a very unlimited number of possible variations. 1) It is possible to control the shape and site of modification of the protein molecule.

One aspect of this invention is the production of modified tRNA molecules, as described below. and the use of this tRNA in producing the desired protein: a) providing a nucleic acid sequence that can be translated into the desired polypeptide; This nucleic acid sequence provides the desired preselected amino acid substitution in the polypeptide. Contain at least one corresponding codon.

b) An amino acid that recognizes the desired codon and polymerizes the amino acid to be substituted into the polypeptide. Obtaining aminoacyl-tRN^ analogs that can function as adapter molecules Rui is to synthesize.

C) Shares a protein translation system containing a nucleic acid sequence and an amino-amplified tRNA analog To do. Here, the protein translation system normally processes nucleic acids except for the following points. performs the function of translating pages. The following points are aminouncle tRNA analogues. However, in other cases, amino acid substitutions are made at locations where naturally occurring amino acids are incorporated. The point is that it can be taken into account.

d) The translation system functions to translate the base sequence, and the translation system also translates the product. Introducing predetermined amino acid analogs at selected codons in proteins be able to exchange.

Although each of these steps is an important part of the invention, the method can be described as follows: Various improvements may be possible for those who are skilled at applying techniques to special uses. cormorant.

Proteins are the basic units that form living organs and perform various functions. typically These proteins have structural support functions, catalytic (enzyme) functions, or acts as a mixture of these two. The protein is menosenger RNA. (■RNA) According to the information contained in the base sequence that forms it, there are 20 common Polypeptide chains are synthesized on bosomes from a set of amino acid monomers. It will be done. mRNA is read in units of three nucleotides (one codon) at a time. Translated by ribosomes. Is it a special sequence At1G or a start codon? The ribosome sequentially reads three nucleotide units to form the “backbone” of translation. Shape.

RNA consists of four types of nucleotides: adenine, /tosine, guanine, and consists of nucleotides containing uranol. The three nucleotide units (codons) are , there are 64 possible combinations, three of which are usually not translated, resulting in Refers to termination of polypeptide chain elongation. These three codons, LIAf:i , [IAA and UGA are normal stop codons. The other 61 codons are each It has an adapter molecule (tRNA) corresponding to each. This tRNA is a message Codons are recognized (or complementarily matched) by complementary sequence matching of bases.

Complementary sequences are contained in these "anticodons" of the tRNA. This t Another part of the RNA adapter molecule is the “extending” protein polypeptide chain. position the amino acid in the correct part of the ribosome so that it acts as a substrate for the reaction Acts as an adapter molecule. Here, the peptide chain that is being synthesized is attached to the amino acid on tRNA. Polymerized to noacylated components. The codon at the 5゛ end is Codes for amino acids. The codon that follows this is the next codon in the peptide chain that is being synthesized. Directs amino acids on the carboxyl side. In this way, the polypeptide chain Synthesized starting from the carboxyl terminal, each successive amino acid Added to the end.

Normally, tRNA enzymatically binds the correct amino acid components with a high degree of fidelity. are combined. Therefore, this adapter molecule is Contains new amino acids. If tRNA receives the wrong amino acid bond, tRNA nevertheless locates the appropriate location of metsenger RNA. recognizes the appropriate anticodon and therefore incorrectly acylated amino acids The components are nevertheless polymerized into the nascent polypeptide chain. add more Therefore, this point is important for tRNAs with modified anticodons that match the stop codon. can also be applied. These are known as "suppressing" tRNAs. because, This is because they suppress the effect of stop codons at appropriate positions on the mRNA.

This phenomenon is, in part, the basic basis of this invention.

This invention provides access to tools and molecules that in many cases have not been chemically specifically defined. use. and general that does not necessarily mean exactly the same meaning in other fields. use terminology. The definitions described below are basically defined on a functional basis.

Many of the techniques and conceptual descriptions used here are from Watson et al. , (1987) Molecular Biology of the Gen e, Vols, 1 and 2. Furthermore, Watson at a l, , Gene is mentioned.

The word "reading" refers to the process by which the protein translation system converts mRNA into A polypeptide that recognizes a given codon and is in the process of synthesizing a special amino acid in the direction of the codon. This is the process of polymerization into the corresponding part of the peptide chain.

The term “misreading” refers to the original protein translation. Codons that correspond to amino acids inserted into polypeptides that are being synthesized in the system is used to mean an incorrect translation of the cipher. (In other words, it's not If so, it is before the aminoacyl-tRNA is incorporated into the protein translation system. Ru. ) The term "stop codon" is used in protein translation systems. A codon used as a signal to stop translation. As a translation system, usually If the codon used as the ``unified code'' is be.

Usually these codons are UGA, UAG and USA. However, It is also possible to create a translation system in which the codons corresponding to amino acids are completely different. So here The term (stop codon) refers to any codon used in a translation system. This means that it includes

“tRNA RNA analog term refers to the transfer of peptide chains during synthesis and codon recognition. Any molecule that is an active tRNA analog . Each tRNA species is not strictly uniform in chemical definition. Because even if it's basic Even if it has little or no effect on the character, there are many This is because many methylations and other modifications or changes in the primary nucleic acid sequence occur. death Therefore, the term refers to a vague core chemical definition that includes various modified forms. Contains righteousness. Normally all tRNA molecules are equipped to recognize special codons. Since it is directly transcribed from a single gene, it is accurately considered a “tRNA.” I can do it. Therefore, a functional definition is much more appropriate than a chemical description. various Many tRNAs from various sources are commonly found according to the base sequence of their “core” backbone. has been defined (Sprin-zel et al, (19g?) Nucle ic　Ac1d　Ra5earch　15:R53;GenBank”/EMB L　Dat≠aank).

On the other hand, the number and location of methylations and other modifications are variable and imprecisely defined. I think there is. This definition includes the following minor modifications of tRNA: It is specifically intended to include each of the variants of heterogeneous or homogeneous tRNA molecular species. be. These subtle modifications include, but are not limited to, methylation changes. Or other modification types, differences in tRNA backbone base sequence (substitutions, additions, deletions, etc.) including base modifications), exogenously derived tRNA, or tRNA molecules generally This includes other molecules that may have related functions. Translation system Phases between components (ribosome, Elonge 7-1 factor, tRNA, etc.) The efficiency of the interaction is not necessarily particularly high. However, the usual techniques If you have this, you will know the basics related to the various specific contents of this invention. You will be able to understand the functions. The minimum requirement for tRNA function is t As RNA is unwrapped through some enzymatic or chemical process, It is necessary that the conjugated molecule has an activity as an acceptor molecule. minutes The kinetics and interaction between children are not particularly critical, but they are important in terms of efficiency. is important.

“The term aminoacyl-tRNA analog refers to aminoacylated tRNA means any of the following analogues of A: a) an adapter factor; function, that is, metsenger RNA, ribosomes, and their associated translation factors. to properly interact with the progenitor and elongenron factor and the polypeptide chain that is being synthesized. thing: and b) including: 1) Properties as a functional anticodon, and il) Polypeptide in the process of synthesis. Amino acid analogue components that can be polymerized into tide chains It should also be noted here that certain methods based on this invention In the method, the amino acid analog must be the terminal amino acid at the end of reading. In that case, polymerization into the polypeptide molecule during synthesis does not necessarily mean that the The fact that amino acids have the ability to additionally accept amino acid monomers (i.e., It does not mean an extension of the peptide.

Chemically, an aminoallated tRNA is defined as a molecule containing: It would be possible to determine: X-A-Y-B-M; Here, X is a nucleotide or a part of the D loop or anticodon loop. Free from modified nucleotides (methylation or other modifications to the base moiety) is the 5' end of tRNA; A is the anticodon part of tRNA ;In the narrow sense, it refers to the 3 nucleotides that are 77 times the codon to be translated; Broadly expanded to include nucleotides near the 3 nucleotides of the anticodon. Y can be a portion of the anticodon loop, variable loop, TφC loop, The nucleotides that make up part of the protein stem and the modified nucleotides (methoxynucleotides) The 3' portion of tRNA consists of ;B is normally not hooded by the tRNA gene, and the 3' tRNA nuclei 5'-pCpC of tRNA added by leotidyl transferase pA-3' end, and M is an amino acid component.

This definition refers to a shortened tRNA molecule with the 3' terminal nucleotide removed, e.g. tRNA (-A), tRNA (-CA) or tRNA (-CCA) ) is also meant to eliminate the possibility of aminoacylation of tRNA molecules such as Note that it is not . If these are functional, they should not be used here. It would be equivalent to a tRNA such as

The term "multi-nucleotide" (MNM) refers to a nucleic acid, typically a ribonucleonucleotide. It refers to a short sequence of cleic acid. This term refers to aminoacyl-tRNA Used in narrowing synthesis. “tRNA(-Z)” is the corresponding tR Shortened by removing short segment 2 from NA (i.e. deacylated form) It also refers to a tRNA molecule with the corresponding Z segment removed.

Alternatively, the truncated tRNA (-Z) can be used as recombinant DNA or can be synthesized by chemical methods.

In a sense, these two segments correspond to multi-nucleotides (MNM). . Thus, the truncated tRNA (-Z) can be attached to a multi-nucleotide molecule (M NM), the tRNA is converted into a deacylated ami/acyl tRNA analog. become a group. One method of this invention involves converting aminoacylated MNM to tRN Used as a substrate to bind to A(-Z) molecules and synthesize aminouncle tRNA analogs. used.

Thus, the term “tRNA(-Z)” refers to aminounruly multi-nucleolyses. Aminoacyl-tRNA analog molecules that have functions by binding to otide It means a molecule that can make . This term specifically refers to the short form of tRNA. Typically, 2 to 3 nucleotides at the 3' nucleotide end are removed. It is intended to be included. Amino unruly multi-nucleo to LRNA(-Z) Aminoal/t has a function by binding to tide (aminoanol-MNM) Synthesize RNA analogs. Typically, 2 and MNM have the same meaning, and the specific Specifically, it is something like 5'-pCpA-3' dinucleotide.

MThe term amino acid analog that does not exist in nature refers to amino acid analogs. or a modified form of that amino acid. This word refers to natural amino acids. modified products, amino acids that do not exist in nature, amino acid analogs, and amino acids that do not exist in nature. Contains noic acid derivatives.

The naturally occurring set of amino acids is the polymerization process normally carried out by ribosomes. Contains amino acids used in the process. These amino acids are usually used in protein polymerization reactions. It has a codon that is /gunar. Even in normal cases, rare forms of proteins There are amino acids, but these are usually one of the 20 commonly used amino acid groups. This is a relatively simple modification of the first one. Furthermore, although it actually occurs in nature, , including amino acids that are not ultimately polymerized during the translation process in their original form. . These natural modifications may affect the polypeptide chain during synthesis or even polypeptide chains (in the case of Apparently results from modification of amino acids after the polymerization reaction that occurs after the completion of the polypeptide chain It is something. These include post-translationally modified amino acids such as 4-hydroxyprolyl 5-hydroxylysine, insulin, and others. These modifications The type of amino acid that is the target of the modification and the type of modification carried out by the extremely limited.

Modified forms of natural amino acids, non-naturally occurring amino acids, amino acid analogs, Amino acid derivatives, etc. are the alpha form of amino acids or have other functions. Means to include all qualifications. This further leads to the substitution of rare forms of atoms. This includes the addition of cofactors and their binding moieties, and the addition of side chains such as glycosylation and acetylation. It is a modification that

The term “preselected codon” refers to the leading sequence of a protein being synthesized. A codon that causes some kind of functional change within a frame. Ru. Therefore, if a particular base sequence has more than one reading frame , this codon needs to affect only one of them.

“Site-specific introduction” involves inserting specific codons into the reading frame of a message. by introducing a specific amino acid analog elsewhere in the polypeptide chain. means to introduce it into a specific part of the body. Codon position and its corresponding amino acid Since the position has a pair of positional correspondences, it is a substitution site for an amino acid analog. is determined by the location of the corresponding codon. as a result, The site specificity of amino acids is determined by the codon location and vice versa. It is.

The polypeptide chain that is being synthesized is translated from the haze RNA 5' upstream of the functional codon. It is an incomplete polypeptide chain that has been The active codon is a point during synthesis. Determine the specificity of the next amino acid analog to be polymerized into the repeptide chain. usually During the polyheftide chain elongation process, the polypeptide chain that is being synthesized is transferred to the ribosome. on the aminoacylated part on the tRNA that recognizes the codon adjacent to the A site of and polymerized.

The words protein and polypeptide actually mean protein or polypeptide. This system can be used to contain products synthesized in this system with functionally equivalent modified molecules. Ru. This is particularly true of proteins, including apoprotein and holoprotein. or polypeptide. The polypeptide molecules included in this definition are: belongs to: a) Adding a modified amino acid to the normal position of the amino acid (corresponding to that amino acid) of replacement; or b) components that resemble amino acids but may differ in structure or composition from amino acids; .

This includes, but is not limited to; i) Beta; Peptidized linkages containing amino groups of gamma, delta or other carbon molecules Components with combinations (i.e., non-alpha amino acids): if > carbons on the polypeptide backbone and atoms other than nitrogen atoms; +i+) (heteroatoms, cyclic or non-cyclic atoms) or metal-binding groups; also correspond to synthetic side chains (including radioisotope specimens) iv) Amino acids containing side chains R such as Amino acids substituted with phonyl, phosphoryl, phosphonyl groups, etc. In addition, there are amino acids with limited φ and φ angles, such as brolin analogs ( A is a dialpha-substituted amino acid.

These methods can basically be applied to any species, especially those with catalytic ability or receptor properties. Basically applicable to the structure and function of enzymes with function or ligand binding ability. It is Noh. Catalytic proteins include, but are not limited to: peptidases, nucleases, glycosidases, and monooxygens. enzymes, dioxygenases, pyridoxulfophosphate-dependent enzymes, flavin-dependent enzymes natural enzymes, lipase and aldolase. Receptor tan/<ri means the following: including, but not limited to, antibodies, T cells, muscarinic receptors, cells, G proteins, lectins, DNA binding proteins, and cytochromes. structure Tannokuri refers to the following: Onn, nruku.

The term “substrate binding site” refers to the tertiary structure of the polypeptide chain that is important for substrate binding. Amino acids that are close to a moiety or are important for its function, or are substrates or ligands. It is an amino acid located near the region where the polypeptide binds to the polypeptide backbone.

The term “enzymatically active site” refers to the positional chemical characteristics necessary for enzyme catalysis. refers to amino acid residues that belong to or are located near the fundamental part of the body. Ru.

The term “protein-protein interface” refers to the interaction between mutually independent polypeptide chains. Residues that are close to the moiety that interacts with each other.

“Cofactor binding” refers to the binding of cofactors and ligands. The matching part or the part used for recognition for that combination, LX, is its neighborhood. It refers to the residue of a part.

Nucleic acid sequence, selected codons, to be translated to create the desired polypeptide chain Needless to say, there are various methods for creating a nucleic acid sequence contained in a specific site. this Methods include, but are not limited to, the following: Use existing sequences, modify natural sequences, synthesize parts or completely new hybrid sequences by combining various naturally occurring sequences and various naturally occurring sequences. Maniatis; Wu and Grossma, such as making tanbak. n, Methods in Enzymology.

Vol, IS3. and Au5ubel et al, (19117) Current Protocols in Mo1ecu attack≠Chichi@Biolo gy.

See Vols, 1 and 2, each of which is specifically included in the reference. thing.

Such sequences include both DNA-type and RNA-type sequences. The DNA type is Incorporated into genes, including but not limited to: sequences incorporated into clo1-extrasomal factors (plasmid, episomal phages, viruses, etc. containing microchromosemes or free-form DNA. and other similar types. Similar RNA types are also included.

The sequence of the RNA to be translated has different codons in different parts of it. will be changed by substitution. Why special instead of different codons? whether one fixed codon is used, and whether each various codon is one of the others. It is not fully understood why it is replaced with . Theoretically, obviously 63 A different amino acid and an additional one amino acid codon and an additional one Translation systems can also be created using stop codons (Watson et al., Gene). Using this method, the universal code is different from all (different genetic codes). It is possible to create a translation with In particular, the starting sequence for making the desired product is It may be a natural sequence, a modified sequence, or a complete (synthetic) sequence.

The location of the substitution may be any one specifically chosen in advance for the insertion of the amino acid. You can also change codons such as What is desirable for use is vague Codons with diversity, i.e. tRNAs selected to complete misreadings. do not choose a codon that would be translated by a different aminoacyl-tRNA. It is.

If there is a truly unique correspondence between codons and aminoacyl-tRNAs, there will be no stoichiometry. Recognizes the codon chosen to make a translation error, although a complete substitution occurs Allows other aminoacyl-tRNAs to act as adapter molecules This may not be stoichiometrically perfect due to some mechanism that causes

Thus, theoretically any codon could be selected for translation errors. However, it is usually used anywhere else in the reading frame of the polypeptide chain. It is common to choose codons that are not known. and the basic translation system used. Not translated by any aminoacyl-tRNA contained within It may be necessary to choose codons.

For these reasons, the stop codon is an in-frame stop codon in the base sequence Kamiike. Not to suit your requirements. Under optimal conditions, it is actually used to stop translation. A stop codon is chosen that is different from the one given. Furthermore, the unique codons selected are It is probably unique because of the power of gene synthesis. To make a specific codon, use the same codon All other parts containing By leaving that special part as a part with a single selected codon, It can be made into a Noh. In addition to this, this system has the same predetermined two or more substitutions with amino acid analogs or two or more substitutions with amino acid analogs; Choose multiple unique codons, such as when using different analogs above. Based on this, two or more permutations can be easily made.

The term “substantially uniform” refers to uniformity at the site of modification and at the dots of the shape. It means the concept of The fact that some modifications are sufficiently uniform means that the vast majority of translations The translation product is homogeneous, typically more than 60% in a single form, preferably 80% It is used to mean that % or more are the same, and in the most optimal case, 98% are the same.

The term “substantially stoichiometric” means that almost all of the products are substituted for the desired moieties. conditions under which this occurs, typically 70 to 80% or more of the product is replaced, preferably 9 This means 0% or more substitution, most optimally 98% or more.

The “protein synthesis system” consists of ribosomes, tRNA, elongation factors, and Addition of mRNA and all the necessary components to translate the mRNA into protein under appropriate conditions. It is a system consisting of elements. This term is also described as 'translation system'.

Typically, all cells have a genetic protein synthesis system. but, It often has very low activity for various reasons. mentioned here In vivo systems are sometimes used for applications in which the necessary amino acids are Difficulties can be encountered in introducing aful-tRNA. This biological system is Conventional microinjection and other mechanisms for introducing exogenous product molecules into cells, e.g. This is possible by using electrical injection into cells. clearly possible technology methods such as polyethylene glycol or Sendai virus fusion. There is a method of fusing cells or ribosomes using Preferred and used translations The system involves microinjection into frog eggs. This microinjection method is similar to other It is also used when using cells as a translation system. More typically, in vitro is preferred. This is because it is a non-naturally occurring amino acid in a highly charged state. This is because it is easy to introduce acyl-tRNA molecules into the synthesis system at high concentrations.

Such translation systems are available for purchase, including Escherichia coli (II: coil), Escel Piae (S, cerevisiae)/wheat germ (wheat g) erm), soluble rabbit cough cell (reticulocyte S) It has been made from things.

One of the characteristics of this method is that it uses aminoacyl-tRNA that does not exist in nature11). This means that different translation systems that can be combined can be performed using the same single piece of information. That is. Different translation systems use different non-naturally occurring amino acids at selected sites. It will be used to import. Thus, a pre-selected create a series of different products, each with an appropriate amino acid inserted that does not exist in nature be able to. This method also applies in other cases to the corresponding amino acid Since it does not have a noacyl-tRNA, this stop codon is a specially selected codon. and use it.

A suitable translation system need not exclude pre-existing tRNAs, but It can be made by adding new tRNA. Unnatural creatures with functions Noacyl-tRNA is synthesized by a combination of the following steps: a) appropriate anticodase and b) select a suitable predetermined amino acid analyte for use; Combine rogues.

Once a mistranslated codon is selected, a tRNA with the corresponding anticodon is Must be selected or composited. Selection preferably separates natural tRNAs. This is done by separating. For manufacturing, mutation and selection or site-specific This may be done by introducing the appropriate anticodon.

The improved translation system does not normally use enzymatic alysis of unnatural adapter molecules. do not have. Therefore, the functional definition of tRNA usually includes enzymatic acylation ability. Not here. However, this point does not necessarily apply when enzymatic acylation is possible. However, this does not exclude that enzymatic acylation is possible when acylation ability is important. It does not exclude.

Attachment of amino acids to tRNA is usually performed using an amino acid tRNA receptor. catalyzed by ze. These enzymes are reversible and the appropriate tRNA (specificity of acylation does not use an anticodon for recognition) and It is highly specific for both amino acids. Sometimes using natural synthetases Although it is possible to improve the specificity of This is not normally done. Therefore, synthesize the appropriate aminoaflu tRNA. It is very important to do so. And since the reaction is reversible, unnatural reactions occur. Minoacyl-tRNA acts as a substrate for some existing synthetase In some way (by mutation or chemical modification) Deactivation/deactivation of lucasynthase by inactivation (e.g. removal by antibodies or other methods) It is important to prevent deacylation activity by removing it with tase. be. Aminoacyl-tRNAs are central to this invention. stop The synthesis of leveraged aminoacyl-tRNA molecules is the main point. Against unnatural amino acids If a synthetase does not exist, this adapter molecule must be created. .

In rare cases, unnatural amino acids can be substrates for synthetases and , bound to the appropriate tRNA. In some other cases, aminoacyl Modifying the amino acid part of tRNA without destroying its adapter function is possible. This method is rarely available. This is because the nucleic acid part This is because the reactive groups usually compete with the specificity of the modification reaction and exclude each other.

An alternative method to synthesize unnatural aminoacyl-tRNA is to synthesize aminoacyl-tRNA. The goal is to synthesize cleotide. And at the same time, this molecule is adjusted to the appropriate tR. and subsequent binding to the NA(-2) molecule. This method works with any kind of amino acid. Quite generally applicable also to acyl-tRNA molecules. What is this aminoacyl-tRNA? , including the usual amino acid bonds. However, compared to the synthetase reaction, Much less efficient. The only limitation is that unnatural amino acids cannot be used in acylation or protection reactions. Do not interfere with the response knob, and also do not interfere with the binding reaction. That is. If so, there are alternative chemical ways to synthesize adapter molecules. Will. The overall idea is to attach unnatural amino acids to oligonucleotides. Then, the nucleotide part is preferably ligated using T4 RNA ligase. It is to be combined with the ocide part. Dinucleotides are preferred for this reaction. desirable, because this is what happens when chemically linking amino acids to nucleotides. , minimize interfering reactions, and then use single nucleotides or AppA analogs. This is because the group has a higher efficiency of coupling. Usually, the 3' end of the tRNA The cleotide is 5'-pCpCpA-3'. Therefore, dinucleotide and The selected one is 5'-pCpA-3'. Other nucleotides (jinuk) leotide or oligonucleotide) such as deoxy-0-ribo A (i.e. pd CpA) or complete deoxyRNA (i.e. DNA) with the 3' end attached to riboA. It would be possible to use what is available. Turn unnatural amino acids into nucleotides After binding, mainly using dinucleotides, tRNA(-Z) is an aminoacyl -multi-nucleotide (aa-MNM) and final aminoacyl t RNA is synthesized.

The fact that the binding reaction is carried out chemically or using an enzymatic reaction means that the binding reaction is carried out chemically or using an enzyme reaction. means an enzyme that has the ability to bind to single-stranded RNA molecules. T4R used in the example Dinucleotides are sufficiently long substrates for NA ligase. Other enzymes are and may require longer or shorter substrates. Sometimes the deprotection reaction is a binding reaction. It may be done after the response.

The tRNA(-Z) material is made from processing natural tRNA. one The method is gene synthesis of tRNA0c'uvn. At this time, natural tRNA ” The anticodon of V is converted into tRNA by aminouncle tRNA synthetase. It can be turned into a termination inhibitor that destroys the recognition of people.

Another method is to generate the suppressor tRNA (-Z) by run-off transcription. That is. In that case, this tRNA is not modified like normal tRNA. stomach. However, it has a certain activity of translation. Each of these tRNA molecules is typically Since it is chemically acylated only once (in contrast to a normal enzymatic reaction), Normally, aminoacyl-tRNAs are used in elongation reactions (with a reduced efficiency). It is often made from.

If suitable tRNA(-Z) can be easily produced in very large quantities by acylation chemistry, If the appropriate tRNA acceptor molecule is modified with high efficiency ( It is very important to use a specially designed system for translation. And this Methods are included to make the materials. Thus, unmodified tRNA Molecules are included in the invention specification. However, in the normal definition of tRNA, is not included.

The chemical method for making aminoacyl-tRNAs can be easily modified from the described method. Ru.

The most obvious is to use a slightly modified tRNA (-Z). it is, a molecule that is somewhat longer, shorter, or modified than the original molecule . These molecules are hereby expressly included in the invention specification. another light A clear improvement is to use mononucleotides, trinucleotides instead of dinucleotides. Alternatively, other oligonucleotides may be used. These are not included in the statement. All are included in the definition of multinucleotide (MNM) molecules. 5'-pC It is possible to use different nucleotide combinations from pA-3'.

to synthesize at least three aminoacyl-dinucleotides (see Figure 1). There are acylation methods used for this purpose. Usually these are reactive groups on dinucleotides. Including blocking. It is recommended to attach the amino acid to the 3' end of the ribose ring. and then removing the blocking group.

The first method uses nitrophenyl sulfen to block the cytidine-based group. It involves treating the dinucleotide with nyl chloride (NFS-CI). 1. This aminoaful NPS was reacted in 1'-carbonidiimidazole (CDI). When this happens, the amino acid binds to the hydroxyl group of the 3' nucleotide ribose ring. Chiosa By treatment with sulfate, the aminoacyl-dinucleotide is left behind. NFS is removed from cytidine.

The second method involves direct synthesis of dinucleotides or dinucleotides treatment with 9-fluorenylmethyloxycarbonyl chloride (FMOCCI), β-one anoethyl chloride (EtCNocl), and Tetrahydropyranyl chloride (THPCI), these are phosphoryl groups , the nucleotide base, and the ribose 2' hydroxyl group. C.D.I. Aminoacyl-2-(4-biphenyl)isopropyloxycarbonyl was reacted in When reacted, it bonds to the 3' hydroxyl group. 1.1.3.3 Tetramethylg Treatment with anidine, 2-pyrinonyldoquine and formic acid removes these protecting groups. is removed and an aminoacyl-dinucleotide is synthesized. aminoacyl vinyl Oxyflucarbonyl (aa-VOC) can be used in place of aminoacyl-BPOC. It may happen.

The third synthetic method is the synthesis of dinucleotides or pendyloxycarbonyl ( Treatment of dinucleotides with CBZ) and tetrahydropyranyl (THP) It is synthesized by protecting the hydroxyl group with the base and ribose 2' and 3'. .

Aminoa/rubenzyloxi7carbonyl is reacted in CDI to form ribose 2' Attach to OH group. When palladium and [1aSO4 are treated in hydrogen and acid, this The protecting group is removed and an amino unrooted nucleotide is produced. carbobenzoki 7 The amino acid is pCNP’! It has been proven that it binds to ipA. , and that NPS and CBZ groups can finally be removed with an efficiency of 35%. has been proven. Enzymes with the ability to acylate nucleotides and contains an enzyme capable of acylating oligonucleotides or tRllA. (Thirsod and K11banov, (1986) J. AtChet Soc, , 108: 5638 & 3977: Sweers and Wong, (1986) J, Am, Chew, @Soc, 108 +6421; 5bav and K11banov, (1987 ) Bioteeh, Bioeng, 29:648; and Ifong et alA, Fluoro- earbObydrateS: ChemiStry&B10ebe+m1Str y, TayIOr (Ed,), ACS sympostum@5eries.

ACS Washington, D, C,).

The selection of predetermined amino acid analogs to be incorporated into a polypeptide Determined by the user's needs. Depending on the user, a number of special modifications may be required. I would like to substitute decorated amino acids in several parts, each with a different purpose. There are cases. In particular, what is of most interest is It is a residue that can modify the properties, activity, or structure of f. of this invention One power of the technology is to overcome the previous limitation that only natural amino acids were the only choice. It has an important possibility of breaking. This invention applies not only to D-amino acids but also to D-amino acids. What kind of things are there, whether they exist naturally or not? However, even one amino acid can be substituted.

Various uses of this invention for proteins include special shapes of residues as described below. , but not limited to, by introducing: a) heavy metal atoms useful for listalography); b) crosswanking agents; C) markers (e.g. radioisotope, spectroscopic, fluorescent, magnetic and electronic); d) Electron acceptors or donors; e ) metal chelator; f) structurally restricted residues; and g) Residues with new base configurations: h) altered acidic or basic residues; i) residues with altered structure (e.g. , homoserine, homocystine or ornithine); and ”) residues with altered hydrogen bonding properties (e.g., amidine vs. amide).

Physical or biological phenomena that can be studied by making substitutions to specific known parts There are many scientific characteristics. Optical markers are used to identify protein and polypeptide complexes. They may be introduced at specific locations in a three-dimensional structure. Inducing residues with different pKa or different residues that affect the pKa of neighboring residues. Introducing residues that have or influence the nucleic acid composition of neighboring residues electron acceptor function, metal chelating function, altered hydrogen bond donor or Receptors, those with modified or restricted bond torsion angles, co-actor bonds or those with special labels for fluorescence or other detection and purification methods, etc. . This invention allows chemical substances beyond the scope of natural amino acid residues to be added to polypeptides. opportunities to escape from the various constraints that have been introduced and broken by nature. provide.

In particular, an important use of this invention is to determine the topological location of a polypeptide. , by binding heavy metal scattering centers, three-dimensional structure of the polypeptide chain occurs during crystallization. This would make it relatively easy to solve the wave equations necessary to determine the next structure. . The structure of a protein is very important and usually depends on how an enzyme performs its function. It becomes a basic property. These functions include catalytic ability, substrate binding specificity, and properties such as the characteristics of the reaction structure and the mechanism of controlled interaction.

However, many methods involve Areas near (near) the location of the protein binding site, protein-protein interface, and coacta binding site. There are some methods that are suitable for evaluating the physical and biochemical properties of parts in close proximity to each other.

In this context, the term close refers to a range of about 50 to 100 ink. If you want to be far away from the area of interest (less than about 30 square meters) , exists in a cubic space of 15 angstroms or less under the most suitable conditions. means.

In vitro translation systems are usually used here. However, sometimes in vivo protein synthesis It is also possible to use a system. A typical in vitro protein synthesis system is a phantom protein synthesis system containing E. coli. from sensory cells and rabbit reticulocyte solubles , the wheat germ solubles, then the yeast solubles, and the various sauce parts. Cells derived from a wide variety of systems are used (Wu and G ross++an, Methods in Enzymology, Vol. 153). A desirable translation system is one with a strong promoter linked to the protein gene. It contains an improved transcription-translation system that has significantly updated transcription under its control. a It is possible to increase the amount of message by adding highly active RNA polymerase. can be increased. Messenger connected to T7 smooth brake motor transcribed by rifampicin-insensitive T7 RNA polymerase, - Universal endogenous transcripts are reversed by inhibiting them in the presence of funvicin. The product of the ground can be lowered. A sustainable translation system is a large scale (Spirin et al., (1999)). 8g) Science 242:1162). Injected into frog eggs and muscle cells By introducing message tRNA or aminoacyl tRNA into cells, It can be introduced inside.

In some cases, one particular translation-based source is desired. desired product The amount produced and subsequent changes depend on the activities present or missing in the translation system. are doing. One such point is glycosylation. , acetylation or other processing enzymes or proteases or other It is a lack of enzymes.

An important advantage of this method for analytical studies is that a large number of permutations of various selected This means that it can be made anywhere. Single with a misread codon When creating an mRNA, an almost unlimited number of different substitutions are made at specific locations. It will be done. The only limitation is the unnatural axes required to be used in the translation and misreading process. Only the number of minoacil-tRNAs. Choosing a stop codon is particularly useful. This is because there is no need to remove or inactivate endogenous tRNA. be. Genetically natural tRNAs are absent for this stop codon, and new A new unnatural amino acid analog can be created simply by adding a new aminoacyl tRN^. It is taken in by doing so. Appropriate tRNA from column 1 of unnatural amino acid analogs In any unnatural amino acid analogue at the chosen location by selecting can also be replaced.

Unnatural amino acids that may be selected include modifications of natural amino acids and non-natural amino acids. Modifications of amino acids, modifications other than α-amino acids (i.e. β, γ, etc.), different steric properties. Amino acids with isomerism (i.e. D-amino acids or asymmetric carbon atoms or (with different stereospecificity of other atoms), amino acid atoms, unusual elements or contains residues and has a coacta binding site.

The simultaneous transcription-translation system allows the synthesized mRNA to be processed without purification or isolation. It involves directly translating the products of a transcription system in the same system. This system initially It is also initiated under the appropriate conditions, which later render the transcript suitable for translation.

Although it is possible to specially design a translation system that has the basic characteristics of a translation system, Alternatively, it is also possible to use a translation system with a highly characteristically modified code. This principle Because of this, the correspondence between codons and amino acids is complete (including different translation systems).

Proteins produced by this method have a variety of properties. For example, a) Uniformity of protein modification site and shape; b) Sufficient stoichiometric modification (for all purposes) and C) Good (known characterization) of the shape and location of the modification.

The numerous possible uses of such products are immediately apparent to protein biochemists. be done. This method can be used to characterize proteins with low background and Simplified by lowering noise due to differently modified shapes. Physical Alternatively, the implications for biochemical analysis may be the availability of purified proteins. or as broad as the types of techniques applied, e.g. EnzyIIology, Vol, l-187; Lehninger, Bi ochemistry;5tryer Biochemis 狽nit凵Fand See Creighton, The Proteins.

For example, when this analysis technique is applied to X-ray crystallography, proteins have a characteristic and uniform weight. Determining the three-dimensional crystal structure of proteins by introducing metal binding centers It will help analyze the waveform pattern data to solve the necessary wave equations (M athews, (1976) Annual Review of Physi cal Chemistry, 27:493-523) B In spectroscopy applications, specific physical properties chosen for unique and uniform locations on proteins By introducing chemical labels, we can identify binding sites, active sites, or other important parts of the electronic structure. Extremely useful for analyzing statically and dynamically well-defined chemical and electronic structures. It is for use.

The following experimental items provide illustrative and non-limiting examples.

experiment Rokumoku and Protein 4 The mutagenesis methodology is based on very well-studied hydrolytic enzymes, as mentioned above. has been advanced using RTEM β-lactamase (Hamilton-Mil ler and Sm1th Eds, (1979) “Beta-Lactam ases'', Academic Press, London; Jack an d 5ykes, @(1971) An n, N, Y, Acad, Sci, 182:243; and Abraham t (1977) J, Antibiot, 30:51; F Amantō■■Chichi@at al.

(19711) Biochemistry17 +2180). of this bacteria (E. coli) The enzyme has a single 29kD chain with one disulfide bond. protein (Sutellffe, (1978) Proc, Nat 1 ．． Acad, Sci, USA75:3737: Ambler and 5c ott, (1978) Proc, matl, Acad, 5et LISA75:3732: and Po1litt and Zalkin, (1983) J, Baetariol, 153:27) @o β-lactama The base sequence of the electron encoding the enzyme has been determined (Sutcliffe, (1978) Proe.

Natl, Acad, Sci, USA75 +3737: Ambrar and 5cott, (1978) Proc, NatlA Aead, S ei, USA75 :3732:and Po1litt and Zalkin, (1983) J, Bacteriol, 153+27), Aβ-lactamases of the same class The three-dimensional structure of has been resolved (Herzberg and Moult, ( 1987) Science 236, 694) and a simple method to measure enzyme activity. There is a method using a spectrophotometer (1 nitrocefin hydrolysis unit (1 μmole Nitrocefin hydrolysis reaction/win/mL, O, 1 mL-trocefin, 5 0mM phosphate buffer, pH 7) corresponds to 0.61 ug of enzyme, It was determined by the Fladford measurement as; o'Callaghan et al, (1972) Antimielob, Ag, Chamoth er, l:283) o This enzyme is Aβ lactam-type antibiotics (penicillins and cephalosporins) with beta-lactamamide conjugates. The compound is inactivated by hydrolysis. The reaction consists of two steps, and serine 7° ( Ser70) is bonded to nucleophilic, and the acyl group - Forms enzyme intermediates. Then the corresponding acid and the free enzyme again (Fisher at al., (1980) Bioch++ 1stry19:2895; Knowles, (1985) Ace, Ch■ Soot@Res, 18: 97: Dalbadie-McFarland et al, (1982 ) Proc, Natl, Aead, Sei, USAV9:6409: and Sigal et al. (1984) J. Biol. Chet 259:532 7).

Phe66 is well conserved in type 4 A β-lactamases (because it Ambler, (1979 Beta-Lactamases"H amilton-Miller and Sm1th Eds, Ac≠р■High Amame@P ress, Nev York pp, 99-125), for the initial mutagenesis. It was chosen as a target because many of the Phenylalani can be easily synthesized by chemical aminoalization reaction. No side chain protection is required at this step. Staphylococcus arales (Σ4 ■ A 25-pressure solution of an enzyme (having 33% homology with this enzyme of E. coli) In the tome crystal structure (1), phenylalanine has an embedded β-tone It exists in a broad loop between the active site and the α-helical domain (H erzberg and Moult, (19B?) Science236: l194) e The structural importance of this residue is that Phe66 is replaced by Ala (pF6 6^) and convert Phe66 to Try (pF66Y) Both mutants were confirmed by making mutants (extremely isolated in crude cell extracts). It showed only slight activity. (All in vitro reactions ζ, E. coli strain JMI OI (△1acpro thi, 5upE, F' traD36.proAB , 1aclQzΔM15).

[C, Janiscb-Perron et al., (1983) Gene2 2:103] o Total in vitro reaction (well, carried out using 5-30 extract [ Pratt, (1984) “Transeription and Tran slation”, Ran5 and Higgins, (Eds,) I RL Press, Oxford Co., this extract (E. coli strain D-10 (r na-10, relAl, 5poT1. metBl) [Ge5teland( 1966) J, Mo1. Biol, 16:67]) Preparation■■ Ta. An attempt was made to purify the enzyme from the F66A mutant, but as a result all activities were lost. The F66Y mutant was purified and analyzed with very low efficiency. It was. Kr+ using F66Y mutant nitrocefin is a wild strain enzyme. It was the same as that of On the other hand, Kcat was 16% of that of the wild strain (the result was Show me (1 (X).

In vitro and in vivo synthesis of β-lactamase was performed using plasmid pSG7. (Figure 3) and was devised considering the following points: RTEMβ- Lactamase is synthesized in vivo with a 23-amino acid Sieder sequence. , and this 1-noder 7-fence allows the enzyme to translocate in the inner membrane. It is removed from the enzyme during the process of generating gen, and the enzyme becomes a fully active enzyme. test tube In order to express the active enzyme in (Kadonaga et al. (1984) J. Biol. Chet 259:2149). This gene (YoI)-dern sequence It lacks a 63 base sequence corresponding to 21 amino acids, making it difficult to express the active enzyme. is sufficient. The truncated gene is driven by a strong hybrid tack promoter. Gene2 placed under transcriptional control (Amann et al. (1983) 5:167), in an in vitro protein synthesis system, the amount of protein synthesized is has been shown to be proportional to the amount of mRNA added to the system (Reine ss and Zubay, (1973) Bioche++, Biophys .

Res, Comm, 53:967). The truncated gene is T7 RNA The φ promoter of bacteriophage T7 was used to complement the reaction by the polymerase. Tabor and Rlchardson, (1985) Proc. Natl, Acad, Sci, LISA82:1974). Ta. This T7 RNA polymerase synthesizes RNA 10 times faster than E. coli polymerase. (Chamberlin and Ryan, (1982) ) The Enzy+*es15:87), Tn903 (Oka at al, @, (1981) J.

Mo1. Biol, 147:217) kanamaynon resistance gene , is inserted on the opposite side of the T7 bromotor and serves as a selection marker for this plasmid. was used.

Protein synthesis requires the addition of an aminouncle suppressor tRNA to the translation apparatus. For ease, the reaction was performed in vitro. Zubay (197 3) Annu, Rev. Gen, 7:267) and Collins (C. Offins, (1979) Gene 6:29) and Pratt, (1979) Gene 6:29). 19g4) “Transcription and Translation” Hanes and 111gg1ns (Eds, ) A IRs Press Some improvements were made by , Oxford, pp, 179-209> In the E. coli complex system, the pH is adjusted to 8.2 to stabilize the afur bond, which is weak against bases. It was used with slight improvements such as lowering it from 7.4 (Figure 4).

The efficiency of synthesis of active β-lactamase initiated by pSG7 is Typically 30-4 per reaction Ice as determined based on yne hydrolysis activity. in the range of 5 μg (see Figure 6). This is 23− per gene copy. This corresponds to 33 copies of the active enzyme. Then, the pBR322-derived psGl field The native Apr promoter control shows an 11-fold increase compared to the constructed enzyme. ( The amount of synthesis in vivo is also different for JMIOI/pSG7 vs. JMIOI/psGI. The ratio is also 11 times calculated based on the specific activity of the crude cell extract. Surprisingly, TR7 RNA polymerase (final concentration of 8500 units/row) ) is added to the reaction solution of plasmid psG1 with T7 bromotor, pSG The production amount of activated enzyme is only 65-70% of the production amount of the reaction solution started in step 7. It was. (A sample of the 434 repressor expressed after a strong tack promoter) In vitro protein synthesis produces more than 150 copies of protein per gene copy. ). β-lactamases synthesized in vitro are precipitated with ammonium sulfate. homogenized by chromatography and anion exchange chromatography. The homogeneity of the protein was determined using SDS polyacrylate (Fig. 4). Determined by lylamide gel electrophoresis. and against nitrocefin. It was assayed whether Keat and KM were the same as those of enzymes synthesized in vivo. . All inhibition reactions were performed using the pSG7 derivative pF66am (Figure 3). Ta. This pF66a garden has Phe66 mutated to TAG.

suppressor tRNA raw used to carry unique amino acids into growing peptide chains on ribosomes The sabre, sir tRNA must satisfy the following two conditions: it is , sufficient amino acids must be inserted in response to the UAG message. Sara What E. coli aminoaflu-tR is present in the in vitro transcription-translation system? It must not be acylated or deacylated even by NA synthetase.

The first condition above does not produce enough protein for purification and further analysis. It is necessary to The second condition is that the protein is inserted into the protein in an in vitro reaction. The only amino acids that are desired are those that do not exist in nature; This is a necessary condition to ensure that none of the amino acids in mel and 5oll (1979) Ann, Rev, Bjoche+* , 48:601: Fersht and Kaethner, (1976) Biochemi Karasho Nitrogen P5:3342: Igoli et al. (1978) Biochemistry 17:3 459:5chreier and Schim+*el (P972) Bio- ch emistryl 1: 1582; and Yarus, (1972) P roc, Natl, Acad, Sei, USA69@:1915) o Yeast Amber suppressor tRNA derived from tRNAPh. and Uhlenbeek (1982) Biochemistry 21:3 921 and 21:855) meets these two requirements based on the following results. It seemed: Residues 34-37 of yeast tRNAPh to 5'-CUAA-3' The replaced yeast tRNA pc’u’+1 is an extended anticod of Yalu. loop hypothesis (Yarus.

(1982) Science 21g+646), a sufficiently efficient suppressor - is expected to be.

In addition to this, Bruce and his collaborators (Miler et al. (1977) J, Mo1. Bi.

+09:275: Bruce et al., (1982) Proc, Natl. , Acad, 5elUSA79nia127; Bossi ashiп@Roth.

(1980) Nature286:123:and Steege, (197 B) Biological Regulation and Development nt”, Vol, 1. Goldberg Ed, Plenum Pres. s) is yeast tRNAPc’u-■ The IIAG codon is fully translated in mammalian protein synthesis systems (weed It was shown that the efficiency was somewhat low in the germ type. fork and co-researchers (in Wok and Wong, (1980) Can, J.B. iochem, 58:213) et al. Setase (PRS) is acylated to E,c　o　i　1　tRNAPh@ In comparison, yeast tRNA was shown to be aminoacylated with an efficiency of less than 1%. did.

East's tRNAPchu- is a combination of Bruce and Bruce a. nd Uhlenbeck.

(1982) Bioehemistry21:855and21:3921) It is produced in milligram quantities by a method that replaces the anticodon loop. This person The method uses modified nucleotides from the anticodon loop of yeast tRNA. Y37 as well as the three anticodon nucleotides G-34, A-35 and A-36. These four removed nucleotides The code is replaced by chemically synthesized CpUpApA, which is an amber suppressor. It has the anticodon sequence required for sir.

Bruce and Uhlenbeck (1 982) Modification of Bjoehemlstry21:855and　21:3921) Details of the improved method are as follows. Perform the steps to remove the brim first. After treatment, tRNA-y was recovered by ethanol precipitation and aniline hydrochloride. Processed by Ride. The resolved product was recovered by ethanol precipitation, Prehalative*-f+U7g PAGE gel (8%, 0.015c+wX 16 e+wX 42cm; 5cg of crude tRNA was added to each gel). Separated. After the bands were stained with 07o2% toluidine blue and cut out, , 2×1o, 10 axis M Na0Ae (pH 4,5) and 1+*M EDT A: Terrorism. 1% 5DSr was eluted. Staining is based on 7E/k and CHCl31C removed by extraction. Half of the tRNA molecules were recovered by ethanol precipitation.

RNase A was concentrated to 2 μg/mL due to partial nuclease degradation. So After that, perform ethanol precipitation, dissolve the precipitate in distilled water, and dissolve phenol and phenol. Le: CHClz (1:1), extracted with CHCl3, precipitated again with ethanol Ta. The ribonucleotide tetramer CpUpApA is a protected nucleoside sequence. -Synthesized by quenchal phosphotriester coupling (Jones et al., (1980) Tetrahedron 36:3015: Boomand Wreestsan, (1984) -01 axis gonucl eotide 5ynthesis'', GaIt Ed,, IRL P Nit ■ Karato B Washington). Completely deprotected CUAA is Takara Shuzo's T4R One half of the RNA molecule was ligated to the 3'-end using NA'J gauze.

The conditions for binding were 50 μM ATP, +9 μM tRNA (half of both present in the reaction solution), 820 gM CUAA and 50 U/mL T4 It is ligase. Binding reactions typically involve 5 mg of RNase A-degraded tRNA. A reaction volume of 10 mL was used. Kinase treatment was with 4 μM tRNA, 1 20gM ATP and 500/mL T4 polynucleotide kinase (R4c hardson (1965) Proc, Natl, Acad, 5ci USA 54:158; and Midgley and Murray ( 1985) EMBOJ, 4:2695). final bond anti The reaction was performed with 25 [1/mL T4 RNA ligase.

Suppressors made using this method have a 3° pCpA amino anchor at one end. The pig is missing its stem. These nucleotides are tRNA RNA repair enzymes. Creotylal transferase can be substituted (Cudny and Deutc) sher, (196g) J, Biol.

Chell, 261:6450), full-length yeast tRNAPchu”+ is obtained. Sabre Nocer tRNA was tested using excess amounts of yeast PR3. In the presence of [''H]-Phe, 30-35% ([3H]-Phe-tRN (calculated based on the radial groove activity incorporated into the A'c'u.n purified product) silized. Misacylation reaction using enzyme (total amount 300 μg) is as follows including. 4 μM tRNAPc’u’a (30 μg, after gel purification) , desalted, lyophilized), 80 μM phenylalanine, 40+* Tris-HCI (pH 8,5), 15mM MgCl2.45μg/mL BSA, 3.3mM DTT, 2++M ATP and 22 units of yeast PR S (This unit is loOpM. Phe is taken in for 2 minutes at 37℃ under the following conditions. Active: 2μM tRNAPh@(Boehringer Mannheim ), 2++M ATP, 3.3mM DTT, 8.1μM Phe, S0mM Na HEPES (pH 7,4), Is + aM MgCl2.25 mM KCl , and 50 μg/■L BSA). The reaction solution was heated to 37°C for 3 minutes. After that, the reaction was carried out by adding 2.5 M Na0Na (pH 4,5) to% V/V. was stopped. Immediately after stopping the reaction, add phenol (0.25M Na0 in advance) Ac, equilibrated at pH 4, 5), phenol: CHCl3 (1:l) , extracted with CHCl3, then precipitated with EtOI+. Extraction and precipitation two more times repeated. The tRNA is then prepared using Pharmacia First Desalting Kara. After desalination in a vacuum, it was lyophilized. Lyophilized acylated tRNA and non-acyl The mixture of conjugated tRNAs was kept at -8 until just before being used in the in vitro protein synthesis reaction. Stored at 0°C.

Under similar reaction conditions, wild-type yeast tRNA"" It is aluminumized up to %. BD-Cellulose F0? Togelafie (ni reig e) tal, (1986) Proc.

Natl, Acad, Sci, USA 83:8604; Ified+ +ann et al,, (19g?) Nature (kondon) 328:830: Johnson et al. (1976) Bioch emistry 15:569; Baldini, et@al.

(1988) Biochemistry 27:7951; Heckler atal, (1984) Tetrahedron@40:87: and l1eckler et al, (1984) Biochemis try 23:146B) acylated tRNA from unacylated tRNA Attempts at separation have resulted in poor resolution and unacceptably low efficiency. However, it was not possible to separate acylated tRNA. Therefore, acylated and unacylated tRN The mixture A was used as it was as an in vitro protein synthesis reaction solution. East P Using RS, tRNA'chu using an analogue of phenylalanine. ・.

Although we have attempted to misacylate the was unsuccessful with various salt and organic solvent concentrations.

Importantly, tRNAPc’u”+ exists in our in vitro system. Not recognized by E. coli aminoacyl-tRNA ancetase (rXJ6) . Using pF66am and a non-acylation suppressor, [”II] Phenylalani As a result of the in vitro reaction initiated in the presence of lactamase, no β-lactamase activity was obtained; Also, when analyzed on a denaturing polyacrylamide gel, the radioactive band of the correct molecular weight was detected. I also couldn't get it. pF66am and [”+1]-Phe-tRNA’chu- In the initiated reaction, the in vitro β-lactamase synthesis reaction from pF66am The bell was 25-20% compared to that from pSG7. These results are that the host's LRNAPchu- meets the intended conditions outlined above. is clear. : tRNA is not enzymatically aminoacylated, and Not even acylated. And it corresponds to acylated amino acids to LIAG It has been inserted.

After purifying yeast LRNAPchu”a (-CA), phenylalanine corresponding to the sequence bound to pCpA (suieh and Noren, unpublished results). A run-off transcript of tRNA was created (Sai+pson and Uh Lenbeck, (1988) Proe, Natl, Acad, Sc i, UrA ll5:1033). This aminoacyl-tRNA was added to an in vitro synthesis reaction. Sometimes β-lactamase activity is increased by substituting the same amount of anticodon loop. β observed when Phe-tRNAPchu- produced by - Efficiency of lactamase activity was 50-65%. run-off suppressor The efficiency of this translation system used is in agreement with the data for tRNA''v synthesized in a similar manner. It is always conveniently compared. (Samuelsson etal, (198g) J. Biol. Chet 263:1392).

-aminoacyl Enzymatic misacylation by aminoaflu-tRNA/ncetase, as described above. is not a common method due to the high specificity of these enzymes. chemical misua Sylation, however, will work for any amino acid-like structure. unharmed Direct chemical acylation of tRNAs requires a large number of reactive sites. It is impractical because it exists on this large molecule. Heeht and collaborators () Ieckler etal, (1984) Tetrahedron 40:87; and Heckler et al, (19g4) Bi och■Takaya Karasonitsu■ 23+1468) chemically converts dinucleotides to create aminoacyl-tRNAs. After acylating dopCp^, it is enzymatically cleaved using T4 RNA ligase. Binding to the 3′ end of the truncated tRNA [tRNA (-CA)] This problem was simplified by Although this method was successful, there are two major problems. suffered from : Since α-amino/protecting group was not removed, aminoacyl jRNA It is limited only to P-site donors, and chemical aminoacylation is very It was the resistivity.

The general methodology for chemical acylation of pCpA is to activation of the carbonyl group followed by the terminal adenosine (the 2° and 3° amru groups are Intersubstitutes quickly in aqueous solution. ) and an ester bond with a diol. a Minoacylation reaction involves the acylation of the exocyclic amine 7 group and the 2° of adenosine. It is complicated because it prefers a 3° nodalization. When α-7 amino group is protected, amino Significantly increases the stability of anruester linkeno against hydrolysis and , inhibits the polymerization reaction during carboxyl group activation (Sehubert and Pinek, (1974) BIochi+aie56:383). but However, the protected group is must be removed in order to be Recently, Brunner (Kreig et al, (1986) Proc, Natl, A cad, Sci, USA 83:8U04: Wiedmann et al, (1987) Nature (Lond on) 328:830: Ohnson et al, A (1976) BiocheIIstry 15:569; Baldini, et al ,, (198g) Biochemistry 27:7X51) The following was shown. Aminoacyl pCpA with α-ami7 group protected LRNA (-CA) without hydrolyzing the amino a/le ester bond. ) could be deprotected before conjugation.

7 mimo of tRNAPc’u- External amines are treated with 0-nitrophenylsulfenyl chloride (NFS-CI). The minimum level of protection is shown. Amino acid α- Amino groups are also protected by NFS groups. NFS-pCpA is used as an activator. using N,N-carbonyldiimidazole with N-blocked Phe. / has been made into a file. The NFS protecting group is removed from the /amine and the amino acid is converted into an aqueous thiosal was produced with very high efficiency using phate (Lapidot et al. , (1970) Biochem, Biophys, Res, Coat 38:559 and He1kkila et al, (1983) A cta, Chet 5cand, B37:857) o Acylation reaction^ The deprotection reaction was performed with an overall efficiency of 14% (according to unpublished results, compared to the model). The main controlling factor for the acylation reaction of the substance was the 2°, 3°-diacylation reaction. As an example, the efficiency of 14% is the same as that of Hecht and Brunne. Favorable compared to the efficiency shown by r, 3-4% (Kreig et a l, (1986) Proc, Natl, Aead, Sci, USA 83:8604; WiedlIIann et al, (19B?) N ature (London) 328:830; Johnson@et a l,,( 1976) Biochemistry 15:569; Baldini, et al., (1988) BiocheIIstry@27:7951: Heckler et al., (1984) Tedrahadron 40 :87; and Heckler at al, (19W4) Bioche IIstry 23:1468). However, Chlade k (Happ et al, (19B?) J.

Org, Che++, 52:5387) recently reported that 5-cpc conducted an aminoacylation reaction of p^ and reported that the final efficiency was 26%. Ta.

The chemical acylation reaction (total volume 80 μL) included the following: : 600BM pCpA-Phe (40 μg), 10 μM tRNA’c’u”a (20 μg, which was purified by gel, desalted, and lyophilized)), 55 *M HEPES (pH 7,5), 2508M ATP, 15mM Ml IC12, 20 mg/mL, DMSO (10% v/v) and 200-ff-ni T4 RNA ligase. The reaction mixture was warmed to 37° C. for 12 minutes; 2. Add 5M Na0Ac (pH 4,5) to 10% V/V to start the reaction. Suspended. However, only one extraction and precipitation was performed. Protected with G-NFS The amyl/acyl pCpA obtained also became a substrate for T4 RNA ligase. but However, it is preferable to deprotect and then conjugate rather than to conjugate with a protective group. Aminoacyl-tRNA with higher efficiency was obtained. Completely deprotected pC pA-Phe was directly ligated to tRNAPc"u"a (- The end was directly excised using the anticodon loop replacement method. (Note that the sable nocer tRNA was synthesized).

From the results of analyzing the uptake of 3H-Phe into purified suppressors, it was found that Phe-t The efficiency of RNAPchu- was 35% (based on analysis by gel electrophoresis). 80 = 90% of tRNAPchu” + (-CA) is LRNAPc with the same mobility (converted to huIIQ). Using this method, tRNAPc’u・11(-CA ) is also D-phenylalanine<D-Phe), (S)-1)-nitrophenyl Lualanine (p-NO2-Phe) + (S)-p-homophenylalanine (2 -Amino-4-phenylbutanoinoquinodo, HPhe), (S)-1)- Fluorophenylalanine (p-FPhe), (S)-3-amino-2-benozyl Propioninoc acid (ABPA) and <5)-2-hydroxy-3-phene Acylated using nylpropionic a/, do (PLA) (in this case α -hydrobin) protecting groups were not used). These aminoacyl-tRNAs are To synthesize mutant β-lactamase using an in vitro protein synthesis reaction system. used (see below). In order to carry out the aminoalization reaction efficiently, acid-labile Protecting groups, protecting groups that can be removed by hydrogenation, or protecting groups of unprotected RNA. Efforts are currently being made to search for non-selective lipases that can be used for aminoacylation. ing. Protecting groups that can be removed by hydrogenation or acid can be used to protect amino acids that do not occur naturally. Protection of the side chains of will also be simplified.

In, pF66a+n and the enzyme enzymatically acylated in the presence of [3H]phenylalanine. Shake 0. (30% aluminized) to a final concentration of 167 μg/mL. In an in vitro reaction initiated using -L was made with an efficiency of 5 μg/l.

This corresponds to 15-20% of the suppression efficiency (Figure 8). I) Using CIIA-Phe Chemically acylated sable nocer (3-aryl/aryl) is 2.8-7. The efficiency was 5 Hg/mL. This is 7-15% of the total from 1+aL of reaction solution. The efficiency is sufficient to purify the enzyme to almost uniformity (Figure 8).

Purification methods include ammonium sulfate precipitation followed by talomadoff Focusing and anion exchange chromatography were used. Importantly, kcst and KM of the produced β-lactamase are the values of the wild-type enzyme produced in vitro. (Figure 10). [3H]-Phe-tRNAPchu・9 The 3H phenylalanine site-specifically inserted into β-lactamase is Confirmed by mapping experiment. Trypsin for TREM β-lactamase There are 27 parts that can be decomposed by, and there are 5 phenylalanine residues. (Sutcliffe, (197B) Proc, Natl, Aca d, Sci USA 75:3737; A+ambler ashiп@5cot t (1978) Proc, Natl, Acad, Sci, USA 75 :3732: Po1litt and Zalkin, i1983) J.

Baeteriol, 153:27). These five phenylalanines are 2 It is distributed in 4 out of 8 tribson degradation fragments. One of these is 8 residues This peptide contains both Phe 66 and Phe 72. β-lacta In the presence of [”lf]phenylalanine, pSG7 was more susceptible to in vitro reactions. more synthesized. The purified radiolabeled enzyme is digested with trypsin, and the fragments are separated using reverse-phase Separated by FPLC (Figure 9). [3H] in RTEM β-lactamase -Four clear radiolabel peaks were observed corresponding to the site of -Phe (Su tcliffe, (1978) Proc, Natl, Acad, Se i USA 7S:3737; Ambler ashi ■ Scott (197B) Proc, Natl, Acad, Sci, LISA 75:3732; Po1litt and Z≠Gekkumiya B (19g3) J, Baeteriol, 153+27: Fisher et al, (198) Biochemistry P9:21195 and Knowles (1985) Ace, Cet Res, 18: 97). The peaks eluted in fractions 44 and 45 are compared to the other three peaks. It has double the count and contains the peptides Ffi6 and F72. it was thought. pF66 in the presence of Phe-tRNA'ellu@+1 Trypson-cleaved peptide from β-lactamase synthesized in vitro from am A similar analysis of Tido shows one radioactive peak. Wild type (pSG7) and suppressed type (pF66am), radioactivity was eluted in fraction 44. The fact is that in wild-type experiments, this peak had twice as much radioactivity as the other peaks. Along with the observation that [3H]-Phe has This strongly suggests that it was inserted at position (F1a).

The phenylalanine analogs D-PHe, Hphe, ABPA and PLA are Each was coupled to a suppressor tRNA as described above. [35S]-Methionine In vitro protein synthesis reactions performed in the presence of p-FPhe, ++-NO Similar levels of radioactivity in the reactions of 2Phe and Hphe As a result, chloroacetic acid (TCA) was incorporated into the precipitate. The smell of these reactions Analysis of the enzymatic reaction rate of β-lactamases synthesized by (Fig. 10). Purified p-N02Phe and Direct quantification of Hphe variants was not possible. This is because both mutant types This is because it was deactivated during attempts at purification. As a result, [35 phantom-methionine Incorporation into TC^ precipitates was used for direct comparative quantification of these variants. It was done. In experiments using D-PHe, PLA and ABPA, β-lactamer No enzyme activity or protein synthesis could be detected.

These results are based on different analyzes of atomic structure and electronic properties in the case of phenylalanine. We show that logs can be substituted into proteins. Replace Phe 6B with tyrosine atomically (4-OH group) and electronically (-NO2 is a good π-electron donor) is different from Phe, and its substitution is performed when kcst is 1. There was a decrease of approximately one-half and no effect on KM. Similarly, Phe Substituting 66 with p-nitrophenylalanine again changes the atomic structure in this case. (4-NO2 group) and electronic structure (-OH group is a good acceptor of π electrons). The result was a 2-fold decrease in kaat and no apparent effect on KM.

On the other hand, when Phe 66 is replaced with p-fluorophenylalanine, the original Physically and electronically, it is similar to Phe, but kc and t increase slightly, but Kn No effect was observed. When Phe 66 is replaced with homophenylalanine, this is electronically similar to Phe, and has a very different atomic structure, but kc is It decreased by about 6 times and rn increased. Both HPhe and p-NO2Phe variants are Significant changes in atomic structure occur in both proteins, but both are unstable and likely proteins. It is thought that protein degradation occurred during the purification process because no unfolding occurred. I was disappointed.

Replacing the amino linkage with an ester linkage (PLA), adding to the backbone methylene group (ABPA) or sterically α-carbon (DP Attempts were made to modify the protein backbone by changing he), but Neither protein synthesis nor enzyme activity was detected. Currently, these results are Protein involvement and stability (PHee is also close to proline residues) are impaired. or that these amino acids function as ribosomal A-site receptors. It is unclear whether this is due to the inability to do so. PLA is polyphenylalanine It has been reported that it binds to the amino terminal position (Herve and Ch. apevjll, (1965) J, Mo1. Biol, 13 Near 57 ), N-acetyl-D-phenylalanine reacts with poly-(U) message. It has also been reported that Roses does not function sufficiently as a P-site donor. ser et al.

(1986) Biochemistry 25:6361 and Heck ler et al., (1983) J. Biol, bhew.

258:4492), Yamane et al. Kdlss of the tertiary complex of -Tu is the tertiary complex of L-Tyr-tRNA-EF-Tu. reported that it was 25 times higher than the combined Kdlss (yamane etat, , (1981) BiocheIlistry 20 Near 059) a EF- Tertiary ■ between Tu and D-Phe-tRNAPchu@+ A similar level of stereochemical selectivity in coalescent formation allows amino acids to be used during in vitro reactions. As well as causing substantial hydrolysis of the full ester, the aminoaflu to the ribosome This results in very low EF-Tu mediated binding to tRNA.

In order to analyze the catalytic constant and specificity of mutant strains, we also used ESR and fluorescence spectroscopy. Mechanical or Sufficient protein can be purified to perform a limited analysis of 7noping. trial In vitro protein synthesis method for tRNA production and tRNA aminountylation reaction Thanks to improvements in chemical chemistry, mutant proteins can be produced in milligram quantities for this purpose. It becomes possible to produce

As mentioned above, this invention provides an improved method of producing engineered proteins. You will be evaluated by doing so. This method is quick, easy, and versatile. There is.

All publications and patent applications mentioned in this specification are of interest to this invention. It shows the technical level of the people (those skilled in the art). All publications and patent applications are located here. Each publication or patent application may be specifically and independently cited and incorporated by reference. Ru. The subject matter of the invention is fully written herein. For people with normal skills, Many changes may be made without departing from the spirit or intended direction of the claims. It is clear that modifications and modifications can be made.

FfG, J.

= −21,5 FIG, -5° FIG, -6° Condolence 1 port, captivated by knowledge, Buhei FIG., J.

UAOfFiG, JO for D-Phe pF6Eiam D-PlrtRNA.

international search report

Claims (42)

[Claims] 1. Site-specific incorporation of non-naturally occurring amino acid analogs into proteins A method for making How to: (a) at least one site in the mRNA sequence encoding said protein; (b) introducing said non-naturally selected codon; and (b) introducing said non-naturally selected codon. amino acid analogues at preselected codons in the polypeptide chain during synthesis. Proteins containing aminoacyl-tRNA analogues that can be polymerized at positions Translating said mRNA sequence in a quality synthesis system. 2. 2. The method according to item 1, wherein the protein synthesis system is an in vitro protein synthesis system. 3. The in vitro protein synthesis system described in paragraph 2 is prepared from the following: Method: a) Escherichia coli; b) Exposure yeast; c) Weed yam; d) Rabbit cough. 4. 2. The method of paragraph 1, wherein the preselected codon is a translation stop codon. 5. According to paragraph 4, the preselected codon is a UGA (amber) codon. the method of. 6. In the fourth section, a preselected codon is inserted at a predetermined site. Method described. 7. A non-naturally occurring amino acid analog is selected from the following groups: The method described in paragraph 1: i) modified natural amino acids; ii) modified uncharged amino acids; iii) modified acidic amino acids; iv) modified basic amino acids; v) non-alpha amino acids; vi) φ, amino acid with φ angle converted; vii) Nitro, amidine, hydroxylamine, quinone, aliphatic compound, cyclic and an amino acid containing a functional group selected from the group of unsaturated chemical groups. 8. Aminoacylated tRHA analogs recognize preselected codons. It is the only aminoacyl-tRNA molecule in the protein synthesis system that can The method according to paragraph 1. 9. Pre-selected codons are used in one of the mRNA sequences that encode a protein. The method according to paragraph 1, wherein the method is introduced into the site. 10. Persons described in paragraph 1 whose protein has a molecular weight of approximately 10,000 Daltons or more Law. 11. Approximately 100 ounces of non-naturally occurring amino acid analogues from the following sites: The method according to paragraph 1, located within the troome: a) a substrate binding site; b) enzyme active site; c) protein-protein interfaces; d) binding sites for cofactors; e) Ligand (agonist or antagonist) binding site. 12. Protein synthesized by the method described in item 1, item 8 or item 10 . 13. protein is fully stoichiometric to one or more predetermined sites. A protein according to item 12 that is substituted. 14. The protein is sufficiently uniformly substituted at one or more predetermined sites. The protein according to item 12, which is 15. The protein according to clause 13, wherein the protein is sufficiently homogeneous in its substitutions. . 16. A method for determining the physical, chemical or biochemical properties of proteins. Therefore, the method is characterized in that it comprises the following steps a) and b): a. ) stoichiometrically at a specific site by the method described in Section 1, Section 8 or Section 10. synthesizing a sufficiently pure protein to be replaced; and b) the protein product. Analyzing physical or biochemical properties. 17. As described in Section 16, the protein is analyzed by X-ray crystallography or NMR. How to put it on. 18. Analysis of the physical or biochemical properties of proteins determines: The method described in paragraph 16: a) Static properties of the polypeptide chain; b) Mechanism of action of enzymatic reactions; c) Specificity of ligand binding of the protein; d) Amino acid of the protein of interest Dynamic interactions of residues with substrates; e) protein folding; f) Interaction of proteins with other proteins, nucleic acids or sugars. 19. Approximately 100 angst of amino acid analogues in which proteins do not occur naturally. 17. The method according to paragraph 16, wherein the method is analyzed in the loam range. 20. The following a), b) are provided, and multiple amino acids are present at preselected amino acid sites in the protein. Method for performing selective permutation of numbers: a) identify sites in the messenger RNA that correspond to preselected amino acid sites; , create a messenger RNA that replaces the mistranslated codon; b) The messenger RNA is combined with two or more aminoacyl-tRNA analogues. The protein is translated using the above translation system, and the protein produced by one translation system is The selected protein can then be processed by other systems at preselected amino acid sites. It is different compared to the protein made. 21. Differences between proteins produced by different translation systems lead to aminoacyl-tRNA by preselecting a bound non-naturally occurring amino acid analogue. 21. The method according to paragraph 20. 22. The protein according to item 20, wherein the protein has a molecular weight of about 10,000 Daltons or more. Method. 23. The method of paragraph 20, wherein one amino acid position is substituted. 24. 20, wherein the non-naturally occurring amino acid substitution is selected from the group: How to put it on. i) d-phenylalanine; ii) (S)-p-nitrophenylalanine; iii) (S)-homopheny Lualanine; iv) (S)-p-fluorophenylalanine; v) (S)-3 -amino-2-benzylpropionic acid; vi) (S)-2-hydroxy-3-ph phenylpropionic acid. 25. The method according to paragraph 20, wherein the codon that is incorrectly translated is a translation stop codon. . 26. 26. The method according to item 25, wherein the translation stop codon is UGA (amber). 27. A method of producing an aminoacyl-tRNA analogue molecule, the method comprising: a method comprising the steps of: a) producing a multinucleotide molecule; 2' or 3' ribosyl hydroxyl part of the 3' terminal nucleotide of (HNM) a preselected non-naturally occurring amino acid molecule by an aminoacylation bond at the position b) an aminoacylated multinucleotide molecule; (aminoacyl-MNM), a tRNA molecule (tRNA(-Z)) with its end truncated ) and having a function in the above aminoacyl-tRNA analogue molecule is formed. 28. A multinucleotide molecule (MNM) is the second molecule corresponding to the 3' end of the tRNA. The method described in Section 7. 29. Clause 27 or where the multinucleotide molecule (MNM) is a dinucleotide; is the method described in Section 28. 30. 30. The method of paragraph 29, wherein the dinucleotide is 5'pCpA3'. 31. The binding of the multinucleotide molecule (MNM) to the tRNA (-Z) molecule is completed. 28. The method of paragraph 27, wherein the entire tRNA molecule is produced. 32. tRHA(-Z) is generated from run-off transcripts The method according to paragraph 27. 33. 2 or 3' terminal nucleotides of a multinucleotide molecule (MNM) Preselected non-naturally occurring amino acids in the 3'ribosyl hydroxy position The method for aminoacylating the analogoda is as follows: The method according to paragraph 27 or 30, characterized in that: a) on the MNM; b) protecting the reactive chemical groups on the amino acid analog with a protecting agent; protecting the minoacylated reactive group with a blocking agent; c) Acylation of MNMs using an amino acid analog protected by a blocking agent to do, d) Removing the protecting or blocking agent from the protected reactive group. 34. The procedure for protecting some or all of the reactive groups is selected from the following group: No. 33, characterized in that the procedure is carried out using a blocking agent or a protective agent. The method described in section: a) 0-nitrophenylsulfenyl (NSP); b) β-cyanoethyl (Et CNO); c) benzyloxycarbonyl (CBZ); d) 9-fluoroenyl Amesyloxycarbonyl (FMOC); e) 2-(4-biphenyl)isopropropylene Pyroxycarbonyl (BPOC); f) Vinyloxycarbonyl (VOC); g) Tetrahydroviranyl (THP); h) Methoxytetrahydroviranyl; i) Photosensitive groups. 35. The protection procedure uses 0-nitrophenyl sal as both a blocking agent and a protecting agent. 34. The method according to paragraph 33, carried out using phenyl (NSP). 36. The binding reaction of aminoacyl-MNM to tRNA (-Z) is a T4RNA ligator. 28. The method of claim 27, wherein the 37. Aminoacyl-tRNA analogoder having the following: a) Formula X-A-Y -M, where: X = 5' nucleotide sequence of the tRNA molecule; A = nucleotide of the anticodon; Y = 3' nucleotide sequence of the tRNA molecule; M = amino acid selected from the following group: Anaroders: i) modified uncharged natural amino acids; ii) modified acidic natural amino acids; iii) non-alpha amino acids; b) Polymerize the M component into the polypeptide chain that is being synthesized, and then proceed with the subsequent peptide polymerization. Activity that controls it to act as a receptor. 38. Item 37, wherein the Y component has the 3' end of 5'-pCpUpA-3' molecule. 39. t aminoacylated by (S)-P-nitrophenylalanine The molecule according to paragraph 38, corresponding to RNApchuoA. here: a) X is 5 of tRNAPchuaA, which includes the D loop and anticodon loop part ’ consists of part; b) A (anticodon) consists of the trinucleotide 5'-pCpUpA-3' ;c) Y is part of the anticodon loop, the variable loop, the TφC loop and the d) M consists of the 3' portion of tRNAPchuoA, including the scepter stem; d) M is (S )-p-nitrophenylalanine. 40. 38. A translation system comprising an aminoacyl-tRNA analogue molecule according to item 37. 41. The aminoacyl-tRNA analog described in item 37 is synthesized by a transcription system. A simultaneous transcription-translation system that is translated by a translation system that includes a reader. 42. Amino acids that do not exist in nature are substituted at specific sites with sufficient stoichiometry. A fully homogeneous protein of approximately 10,000 Daltons or more.