TW381178B - Compositions and methods for protein structural determinations - Google Patents

Abstract

A method for determining three-dimensional structural information of a protein involves producing the protein in a form substantially labeled with C13 or N15 or both or substantially labeled with N15 and C13 and partially labeled with H2 and subjecting the protein to nuclear magnetic resonance spectroscopic analysis. The isotopically labeled protein is produced by a method which involves producing a substantially labeled microbial protein hydrolysate, subjecting the protein hydrolysate to cation exchange chromatography to produce a partially purified labeled amino acid mixture, subjecting the partially purified labeled amino acid mixture to anion exchange chromatography to produce a purified labeled amino acid mixture and supplementing the purified labeled amino acid mixture with isotopically labeled cysteine and optionally with isotopically labeled glutamine and asparagine.

Description

A7

V. Description of the Invention (/) Invention Gion (Please read the notes on the back before filling this page) The present invention relates to the determination of the three-dimensional structure of biological macromolecules, especially for proteins. In particular, it relates to novel novel compounds and methods for measuring the three-dimensional structure of proteins expressed by cultured mammalian cells or insect cells using N-spectroscopy. Invented over the years. * For the determination of k-dimensional structure of biological macromolecules, especially proteins, there is great interest. The so-called "structure-function" research has discussed the structure and biological activity of certain molecules, or certain types of molecules, due to the pioneering projects of structure exploration by Nobel laureates, such as Perutz and his companions. Discussion on the structure of heme (perutz, M.F · eta 1., LaJLU.C-.e ...., LSi. 416-422 (1960)) and the study of DNA structure by Watson and Crick (Watson, JD , And Crick, F · H. C ·, N_a..t.nre, 111, 737 (1953)), make structural studies of great importance in biological sciences. Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs, the concept of “rational drug design” has recently been derived. This drug design strategy is about specifying a specific biomolecule, the "active part a *" of its three-dimensional structure, such as a protein. This biomolecule can be a receptor, enzyme, hormone, or other biologically active molecule. Knowing the active area The three-dimensional structure allows scientists to design molecules that can inhibit, mimic or enhance the natural biological activity of the molecule. (Appelt, K., et al., J. MpH. Π h p. M.. 3 4. 1925 (1991)). Therefore, the determination of the three-dimensional structure of biomolecules has great practicality and commercial significance. Firstly, the technique of measuring the three-dimensional structure using K was developed as X-ray crystallography. The structures of heme and DNA were determined by this technique. X-ray crystallography is to apply this paper size to the Chinese National Standard (CNS) / 4 specification (210X297 mm) 83. 3. 10,000 printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs Α7 Β7 V. Description of the invention (> ) The crystalline KX-rays of the test object are caused by the atomic refraction of regular molecules in the crystal. The scattered X-rays are captured by the photographic plate. Then the K-scaling technique is used to develop the image. This diffraction X- Rays A series of dot images on the plate can be used to define the crystal structure of the molecule. For large molecules, K heavy metal ions, such as ruthenium, must be used to crystallize them, and κ to avoid uncertainty caused by phase differences. Recently, another technique, nuclear magnetic resonance ("N MR") spectroscopy, has been developed to determine the three-dimensional structure of biomolecules, especially proteins. NMR spectroscopy was originally developed in the 1950s and is used to analyze small compounds An effective step in the structure is the same as analyzing a molecular weight of $ 1000 daltons. In short, this technology puts a substance (usually stored in a suitable tincture) in a strong magnetic field. Although M powerful telecommunications excite the nuclei of different atoms, Arrange according to the magnetic field until K Telecom adds energy. It absorbs this energy and radiates it again at a certain frequency (resonance). It is based on the chemical environment of the nucleus i) and nucleus nucleus (mostly determined by the bonding). What's more, resonance can be transmitted from one nucleus to another via a bond, or in a three-dimensional space, providing a specific nucleus, its surrounding environment and its neighboring atoms. Nuclear information. However, it is important to note that not all nuclei have NMR activity. In fact, not all isotopes of the same element are active. For example, although "normal" hydrogen, 1 has NMR activity, but , Deuterium (deuterium), 2 Η, is not active. Therefore, any substance containing 1 Η hydrogen under normal circumstances can be made invisible in the hydrogen N MR spectrum by replacing all 1 Η hydrogen K 2 Η ". Therefore, the NMR spectrum of water-soluble substances is determined in a 2 Η 20 solution. * Μ signal to avoid water. In contrast," normal "carbon 1 2 C does not have NMR activity, and its stable isotope λ -4- (Please read the notes on the back before filling this page)

This paper size applies Chinese National Standards (CNS), 4 specifications (210 × 297 mm) 83. 3.! 〇, 〇〇〇 Consumer Standards of the Central Standards Bureau of the Ministry of Economic Affairs printed A7 B7 V. Description of the invention (>) 13C , Which accounts for about 1% of natural carbon, but has NMR activity. Similarly, "normal" nitrogen, 14N, does not have NMR activity, and its stable isotope 1SN, similarly, it occupies about 1% of natural nitrogen and is active. It is known that for small molecules, if the experiment is performed with a sufficient amount of substance and sufficient time, this low natural inventory is sufficient to produce the data required for the experiment. With the advancement of both hardware and software, the size of molecules that can be analyzed by the above technology has increased to about 10,000, 0 0 0 d a 11 ο n s, which is the size of small proteins. Therefore, the determination of protein structure using NMR spectroscopy has only begun a few years ago. It is quickly understood that the size limitation can be overcome by replacing 14K and 12C isotopes without HMR activity in the protein, and stable isotopes 15K and 1C with HMR activity in the protein. One way to achieve this substitution is to grow microorganisms that can produce this protein in a medium labeled with this isotope. In the past 2 or 3 years, the 1SN-labeled and 13C-labeled proteins have increased the size limit that can be analyzed to about 15kd and 25kd, respectively. The completion of this isotope replacement method is to genetically engineer * bacteria or yeasts that can produce the selected protein to grow in a medium containing and / or 1-marked --- substance. In fact, this medium usually contains 1SC-labeled glucose and / or 1-labeled ammonium salts. (Kay, L. et al., S P. i P · η PP. 249. 411 (1990) and listed references) ° Recently, there have been reports of the use of labeled protein hydrolysates as bacteria and Yeast nutrient medium. See International Patent Applications, Publication Η〇 · 90/15525 * Published December 27, 1990. When the 13C and 15N labels make the NMR structure determination of the protein larger than the foregoing, the use of this technique to analyze proteins higher than 25 kd yields ambiguous paper standards applicable to Chinese national standards (CNS IA4 specifications (21 0X 297 mm) ) 83. 3. 10,000 (Please read the precautions on the back before filling out this page) 0 ^ ------ 1T ------ β Employees ’Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs A7 B7 V. Invention Explain (仏) the result. At this size * resonances from different atoms are too broad to be resolved. It was pointed out in a recent Yidu report that using triple-labeling, i.e. partially bound heavy hydrogen, 2 Europium, M, and 1 3 C and 15 N isotopes can reduce the signal scattered in larger molecules. Bax,, 1. Am. Chem.

Soc., LL5_: 4369 (1993). Therefore, NMR is used to determine the protein structure above 25 kd, and it is better to use triple labeling medium to prepare its labeled protein. For bacterial proteins, the bacteria can be cultured in a mixture containing H20 and 2H20 to obtain a portion of the 2H-label. However, this method is not sufficient to produce mammalian cell proteins that are appropriately labeled. Therefore, there are certain restrictions on the composition and method for MR structure measurement. Many proteins whose structure and function are to be explored come from mammals. What's more, the proteins to be explored in rational human drug design are actually from mammals, e. Humans. Therefore, X-ray crystallography or NMR spectroscopy cannot be widely used to detect proteins produced from mammalian cells. X-ray crystallography, by definition, requires crystals, but mammalian cells are extremely difficult to crystallize. To date, only a few antibodies and receptors derived from mammalian cells have produced patterns suitable for crystallographic analysis. ---- These crystals * are usually selected from a part of the molecule. When reviewing the data derived from the molecular segment, one must be careful, because it is not known whether this part of the main molecule, when it exists alone, will still have the same structure as that in the intact molecule. In particular, X-ray crystallography cannot be applied to substances where crystals cannot be obtained. NM structural studies are limited to the need to express labeled proteins in bacteria or yeast. However, most mammalian proteins contain important post-translational modifications * which cannot be achieved in bacterial and yeast systems. That is -6-This paper size applies to Chinese National Standards (CNS), 4 specifications (210 × 297 mm) 83. 3. 10,000 (Please read the precautions on the back before filling this page)

The consumer cooperation of the Central Economic and Technical Bureau of the Ministry of Economic Affairs of the People's Republic of China Du printed A7 B7 5. The invention description (Γ) states that, after being properly folded and interactively bonded with a disulfide bond *, it can be connected to oligosaccharide branches or can be hydrolyzed by proteins After excision, an active state is generated. Proteins produced by bacteria or yeasts generally do not possess the biological activity of proteins produced by mammalian cells. In fact, in some cases * mammalian proteins cannot be produced by bacteria. For the above reasons, in the mid-1980s, the biotechnology industry will produce recombinant therapeutic proteins, such as histone cytosinogen activating factor 'factor vm: c * erythropoietin, etc., from bacterial performance to mammalian performance. Some For mammalian cell proteins, the genes to be explored for the molecular part are cloned into bacteria and allowed to grow on the isotopically labeled medium Φ, and the isotopically labeled fragments are obtained for KHMR analysis. Similarly, only molecules that can be expressed in bacteria can be used for discussion. (E.q. See Driscoll, P.C., et al., Nature, 353, October 24, 199 1). Due to the lack of post-translational modifications in the bacterial expression system, the resulting molecule to be inspected lacks this post-translational modification, such as glycosylation etc., and, similarly, one suspects the value of the resulting structure. As with X-ray crystallography, it is constantly questioning the value of the structure of the resulting protein fragments. -------- At present, mammalian cells and insect cells have been developed as host-carrier systems. Mammalian cell lines, such as Chinese golden rat ovary (CH0) cells, COS cells, and insect cell lines, such as SD nd opp ΓΑ · cell lines SF9 and SF 21 (Luckow, Vi \. And Summers, MD, Biot p. h η n 1 ngv. g_47-55 (1988)) have all been found to post-translationally modify the produced mammalian proteins to make them similar to natural proteins. Use NMR to study proteins produced by mammalian cells and insect cells. This paper is sized to the Chinese National Standard (CNS) _A4 (210X 297 mm) 83.3.! 0, 〇〇〇〇nn n ^ n nflfl — m ^ i Km ^ \\ T / · t ^ itf nnf, ^ ¾ i (Please read the precautions on the back before filling this page) Printed by the Consumers' Cooperative of the China Standards Bureau, Ministry of Economic Affairs, printed A7 ___: _-- ~~ — V. Description of the invention (&Amp;) It has only a certain effect, which is the same as the method used for bacteria, but it takes the isotopes such as 13c or 13C and 15N as analogs. However, bacteria can grow in a simple mixture of glucose and salt, and mammalian and insect cells require all amino acids other than glucose for growth. For example, in order to obtain a protein completely labeled by 13c and / or 1 s N from mammalian cells, all necessary amino acids must be present, and all must be completely labeled by 13 C and / or 15 N. A theoretical method for producing isotope-labeled media is to use isotope-labeled protein hydrolysates. Unfortunately, when hydrolyzing proteins to form amino acids, by-products are also formed, which are toxic to mammalian cells. Use of unpurified hydrolysate * will result in rapid cell death. What's more, traditional hydrolysis steps will destroy certain essential amino acids *, and currently, methods to avoid such damage often cause toxicity. On the other hand * techniques are known for separating and deactivating a single amino acid. For example, LeMaster and colleagues published a report (An ^ l. Biochem., 1_2_2_, 218 (1982)), which describes methods of purification and amino acids. It only requires less than 5 column chromatography purification steps, and although fully labeled cysteine and glutamine cannot be separated, the tryptophan obtained is "unstable". The three amino acids mentioned above are all required for the growth of mammalian and insect cells that serve as hosts for the production of heavy protein. Furthermore, this step uses hexahydropyridine as a primer, so that the amino acid is extracted from the preparative chromatography column. Hexazine is known to be highly toxic and a regulated substance. Therefore, the steps for purifying a single amino acid are complicated, time consuming *, and low yields, and therefore, are not economical. Therefore, although some i3C and / or 1 5 N amino acids are commercially available, there are only a few, and most of them are not.-8-This paper applies the Chinese National Standard (CNS) Standard 4 (210X297 mm) I 83. 3. 10,000 (Please read the notes on the back before filling this page)

A7 B7 printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs 5. Description of the invention (77) Recently, Fesik and his colleagues published a method that can be used to produce isotopically labeled proteins obtained from mammalian cells, which are used in NMR structures Research (B i ο nh ρ. M ί s t. R V. 3 1. Ho. 51, 12713, (1992)). In the presence of tryptamine and oxazole, K-formamic acid hydrolyzes algae and bacterial proteins labeled with isotopes. The latter reagents are used for the purpose of "suicide base". M reduces tryptophan and weaving acid respectively. The hydrolysate M LeMaster and his colleagues were purified according to the procedure described; that is, the hydrolysate was added to the H + -type cation exchange resin, and M hexahydropyridine was used to pour out the amino group K in a group. The amino acid-containing portion was mixed, evaporated to dryness, and then dissolved in water. The sodium hydroxide was adjusted to 11.5, and the resulting solution was evaporated to maintain a fixed state. "Means that there is no more ammonia or hexahydropyridine. Removed. " This amino acid K molecular weight 500 membrane filter was passed through a filter to remove impurities, and then freeze-dried. The author did not indicate that the resulting amino acid can be directly used at U.e. at high pH) * or M acid to neutralize the output of the solution. The work of Fesik et al., Although showing technological advances, has not been able to provide fully labeled mammalian cell expression proteins that can be used for NMR structure determination. First, the hydrolysis conditions used destroyed asparagine, glutamine, and cysteine, leaving only "very small amounts" of tryptophan on page 27715 (Table 1). Second, this step uses hexahydropyridine as the eluent. As mentioned above, it is toxic and a regulated substance. Third, LeMaster stated in its original report that one of the "suicide bases", oxazole will be co-produced with the amino acid leucine. LeMaster uses a crystallization reaction with leucine to remove hoofs. I7 e s ik et al did not mention this' crystallization step, and in fact, it was not possible to perform this step because a single amino acid was not isolated in the work of Fes ik et al. This paper size applies to Chinese National Standards (CNS) .. A4 size (210X297 mm) 83. 3. 10,000 (Please read the notes on the back before filling out this page),-»1 & Employees' Cooperatives, Central Bureau of Standards, Ministry of Economic Affairs Printing A7 B7 V. Description of the invention (and). Fesik et al. Described the use of increasing the output value of the solution to 11.5 'after heating to volatilize until the pH value stabilized, to remove the hexapyridine eluate. Here, especially at the high temperature (boiling point 106 ° c) at which hexahydropyridine is removed, it is very likely that a racemic reaction will occur and / or the amino acid will be violated by the hexahydropyridine / sodium hydroxide mixture to form a nucleophilic reaction. . Such reactions will reduce the amount of available amino acids in the mixture and reduce their effectiveness as a growth medium. What's more, the author himself understands that the heating volatilization step will stop when the stable solution pH indicates that "ammonia or hexahydropyridine d is completely removed". Therefore, the amino acid mixture may contain a small amount of highly toxic hexahydropyridine. More importantly, it lacks asparagine, glutamine, and cysteine, and contains only "very small amounts" of tryptophan (page 1 27 1 5; Table 1). Although the lack of asparagine is not important in the system tested by Fesik et al., Glutamine is extremely important for cell growth (12716, Fig. 2). The authors provide an enzyme method for synthesizing glutamine with glutamic acid. Therefore, for this reaction to be valuable, it must have appropriately labeled glutamine. As the author points out, 13C, 1SN labeled glutamic acid is available. However, triple labeled glutamic acid is not available. -------- Correspondingly, Fesik does not provide any method for preparing labeled cysteine. Only 1SN labeled cysteine is commercially available. Cannot get double labeled 13C, cysteine or triple labeled 2H, 13C, 15N-cysteine. Therefore, the method adopted by Fesik and his colleagues, except for the simple 15N-labeling, cannot obtain the finished product, because cysteine and tryptophan cannot be properly labeled. What's more, through cell metabolism, incorrectly labeled cysteine can be transferred to other amino acids, resulting in missed isotopes. -10-This paper size applies Chinese National Standard (CNS), Α4 size (210X297mm) 83. 3. 10,000. (Please read the precautions on the back before filling this page), and install. 、 \ = ° Α7 Β7 5 2. Description of the invention (^) In principle, the easiest way to produce labeled cysteine, including triple labeling *, is to cultivate an organism in a suitably labeled cysteine-rich medium *, and from this organism The protein is isolated from cysteine. This organism is similar to the known technology and includes purple sulfur bacteria such as Phodopseudomonas soRroiries and c 3 ds ij only tp »and other homocysteine organisms such as L aptnthrixdisf; ndh 〇 ra and Sc..faJ.zop.hy_LljLiii commune, and bacteria that produce homocysteic acid protein by using recombinant M, such as ATCC 31448 * and E. coli that regulates the human insulin A bond by M. Hemicarbine Μ hydrolysis method, obtained from protein separation. The only method known to hydrolyze protein with K without concomitant damage to cysteine is hydrolysis under alkaline conditions (see Okuda, Pr. Acad. Tokyo. Z_ , 2 7 7) And ί i) K enzyme hydrolysis. Unfortunately, neither of the above two methods is suitable for producing labeled cysteine, especially triple labeling. Hydrolysis under alkaline conditions * will result in the required L-half Cysteine is a racemic reaction, and it also causes a variety of other useful, isotope-labeled amino acids to be destroyed. Enzymatic hydrolysis can easily cause isotope pollution caused by enzyme decomposition products, especially long-term hydrolysis reactions. In the mixture used by Fesik et al., The "very small amount" of tryptophan accompanying cysteine is not sufficient for cell growth without additional addition. (Page 12715, Table 1; Page 12716 (Fig 1 and 2). Although 15H-labeled tryptophan can be purchased, 15N and triple labeling are not available. Therefore, in addition to labeling, tryptophan is used as an additive in the labeling experiment. , Will cause the same problem as the lack of proper labeling of cysteine, that is, incomplete isotope labeling. ^ 11-This paper size applies to the Chinese national standard (CNS M4 specification (210X 297 mm))

Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 83. 3. 10,000 (Please read the precautions on the back before filling this page)

Printed by the staff of the Central Economic and Technical Bureau of the Ministry of Economic Affairs of the People's Republic of China Du printed A7 B7 V. Description of the invention (/ ο) Therefore, the method provided by Fesik and his colleagues can only obtain the complete isotope-labeled protein among the labeled proteins. Although this method is a more advanced method, as mentioned above, 15N-labeled amino acids are already commercially available. In the 13c '15b labeling experiment, cysteine and tryptophan cannot be fully labeled *, and in the 2H-labeling experiment and triple labeling test, the cysteine * tryptophan and glutamine cannot be correctly labeled Mark. Another disadvantage of using the proteolytic step to prepare mammalian cell culture media is that under the conditions of water I #, the amino acids • lipidamine and asparagine that appear in the starting protein can be converted into bran respectively. Amino acid and aspartic acid. Most mammalian cell culture media contain a small amount of glutamine and a portion of glutamine. This medium has been developed to produce mammalian cells with appropriate performance *, ie its cell viability and productivity. In fact, almost all mammalian cells produced by using M as a protein for NMR analysis are cultured in a medium containing a small amount of glutamic acid and a high amount of glutamine. In order to be applicable to the widest range of cell lines, K is used as a culture medium for mammalian cell isotope-labeled proteins, which preferably contains a small amount of glutamic acid and most of glutamine. Similarly, it is preferred that the culture medium contains a large part of aspartame * and almost no aspartic acid. Therefore, there is a need for a method to specifically remove glutamic acid and / or aspartic acid from an amino acid mixture without changing the proportion of other amino acids present, and to obtain the obtained glutamic acid and / Or aspartic acid is converted to appropriately labeled glutamine and / or aspartamine, and the resulting glutamine and / or aspartamine is added to the amino acid mixture. Currently, there is no specific method to specifically remove glutamic acid from amino acid trickle. Has been published to use enzyme method to convert glutamic acid to gluten-12-l · —, ---.— G Pack— (Please read the precautions on the back before filling this page) Order .. — @ --- 本Paper size applies to Chinese national standards (CNS specifications (210X297 mm) 83. 3. 10,000 Consumers 'cooperation with the Central Bureau of Standards of the Ministry of Economic Affairs Du printed A7 I 1 ____' _B7__ V. Description of the invention (//). Linamine (Fesik , Et al., Biochemistry. 31 (51), 12713 (1992)). Unfortunately, this reaction is extremely slow (3-4 days), and accompanied by enzyme cleavage, it will produce natural amino acid pollution. More In addition, the triple labeling of amino acids, the partial labeling of i.e. 2 及 and the complete M 1 3 C and 1 s N labeling, the occurrence of atoms can slow down the conversion of glutamic acid to gluten by the 2H label. The enzyme reaction of amines, coupled with the 2H isotope effect. Finally, there is currently no enzyme method to convert asparagus to asparagine.

At present, a report (Biochemistry · 3 丄 (51) 12778 (1992)) published by Hsu and Armitage on the structure of the binding of the immunosuppressive drug CyCI0Sporin A to cyclophilin by K NMR. The isotope 2Η, which has no NMR activity, is labeled cyclophiHn * and is produced by bacteria. They can therefore study the structure of the cyclosporin A / cyclophilin complex * without interference from cyclophilin messages. Due to the importance of mammalian partner / receptor interactions, mammalian cell proteins, especially the receptor, M 2 Η, need to be fully labeled. Nutrient bases of labeled mammalian cells that can achieve this goal are still not available. 1- For the above reasons, for general structure-function research and rational drug design, a fully labeled composition and method is required, which uses K to define the three-dimensional structure of mammalian cell proteins and protein complexes. Therefore, it is necessary to produce mammalian cell cytosolic proteins with fully stabilized K isotopes. Hair removal according to the present invention. The step of determining the three-dimensional structure of a protein according to the present invention includes growing mammalian or insect cells capable of producing a protein to be explored in a nutrient medium under protein-producing conditions. -13-This paper size applies to China National Standards (CNS). _Λ4 size (210X297 mm> 83.3.10,000 (Please read the precautions on the back before filling this page)

A7 I l · _B7 printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs 5. Description of the invention ((>). Sources of amino acids and carbohydrates for long-term use must have mineral values and growth factors, of which are used for cells Synthetic amino acids and other substrates are labeled with HMR-active isotopes; (b) the labeled protein is separated from the nutrient medium and its labeled state and < c) the protein is analyzed by NMR spectroscopy K defines its three-dimensional structure. In another aspect of the present invention, the steps of the method for determining the three-dimensional structure of a protein include growing mammalian or insect cells capable of producing a protein to be explored in a nutrient medium under conditions of white matter-production, and the medium contains all growth Sources of amino acids and carbohydrates * must have mineral values and growth factors, which are nutritional. The carbon atoms of all amino acids and carbohydrate sources in the medium are 13c; (b > Isolate the labeled protein from the nutrient medium Moreover, the three-dimensional structure of the protein is determined by the labeled state and the NMR spectrum analysis of the protein. In another aspect of the present invention, the steps of the method for determining the three-dimensional structure of the protein, including the step of protein-production conditions, can produce To explore the growth of protein mammalian or insect cells in a nutrient medium, this medium contains all amino acids and carbohydrate sources for growth, must be minerals and azole growth factor, in which the nutrient medium The carbon atom of all amino acids and carbohydrate sources is 13C, and the nitrogen atom of its amino acid is lsN; < b) by The labeled protein * is isolated from the nutrient medium and its labeled state is analyzed. This protein is subjected to NMR spectral analysis to determine its three-dimensional structure. On the other hand, the present invention is to define the three-dimensional structure of the first molecule and form a complex with the second molecule, at least one of which is a protein. This method is to label the first molecule K with NMR-active isotopes, and M deuterium to label the second molecule *, and make the first. ≪ 〇 »-14-This paper size applies Chinese National Standard (CNS ) / Α4 specifications (210 × 297 mm) _ ~~~ 83. 3. 10,000 (Please read the notes on the back first, and then fill out this page)

A7 B7 V. Description of the invention (1 ^ 7) ~ The first and second molecules form a complex, and this complex is then subjected to NMR spectral analysis * K defines the three-dimensional structure of the first molecule. Another aspect of the invention is a novel nutrient medium that can supply mammalian or insect cell growth * which contains all the amino acids, carbohydrate sources, and essential minerals and growth factors required for cell growth, of which * All amino acids and other matrices used in the synthesis of proteins by cells are marked with M 13C or M Ύ. The present invention also includes a method for producing a fully isotopically labeled amino acid mixture, which comprises: 微生物 growing microorganisms in a nutrient medium to grow * wherein all carbon sources used as protein biosynthetic substrates are 13 C; (b > by The protein was recovered from this microbial culture liquid; (0 in the presence of sulfhydryl-based selective agent, K • _, non-oxidative conditions hydrolyze the protein * K to produce an amino acid mixture; @This unpurified amino group The acid mixture is added to the cation exchange resin to produce a partially purified amino acid mixture; and then the partially deactivated amino acid mixture is added to the anion exchange resin to produce a purified amino acid mixture; (f > Human foot. Amount of 13C-labeled cysteine into the purified amino acid mixture, so that foot κ can be supplied to mammalian and insect cells for protein production. The above method can also be used to produce, 15M-double labeled Amino acid mixtures, or 2H, 13C, 15N-triple labeled amino acids, or printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs to produce 2H-labeled amino acid mixtures. It also provides a method for converting glutamic acid and aspartic acid to glutamine and aspartic acid, respectively, and provides an isotope-labeled mammalian cell culture medium, which contains a small amount of glutamic acid and a high amount of bran. Amidine, and it optionally contains very little or no aspartic acid and a high amount of aspartamine. In particular, the present invention provides a specific method, which can be bran-15-83. 3. 10,000 (Please read the notes on the back before filling in this page) This paper size applies to Chinese national standard (CNS LA4 specification (210X297 mm) A7

V. Description of the invention (/ J) Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs Amino acid is removed from the amino acid mixture produced by the above method, and the κ chemical method is used to convert glutamic acid to glutamine. Using the same procedure, aspartic acid can be synthesized from aspartic acid by the κ chemical method. This method will not affect its efficiency due to the isotope labeling, including 2H ’. The present invention further provides a method for isolating cysteine labeled with an isotope from a labeled docetaxel and an amino acid mixture. This method can be used to produce K, 1 3c and 1 5N cysteine as any sister label. This also includes the addition of amino acid mixtures labeled with cysteic acid. The invention provides a method for determining the three-dimensional structure of a protein produced by mammalian or insect cells. Since mammalian or insect cells are used as hosts for recombinant DNA, they can produce proteins that are similar to or the same as their natural three-dimensional structure. Therefore, the present invention can provide a technique to use K to explore the biological-active protein * structure-function relationship. The steps include culturing mammalian hermaphrodites in a nutrient medium, in which all amino acids are completely labeled with M—or multiple isotopes with NMR activity. In the present invention, the protein is triple-labeled with K13C, or both, even M2H, 13C and 1SH. The present invention further provides a method whereby the protein is only fully labeled, making it non-NMR active. The latter is particularly effective when exploring the molecular structure of complexes, such as hormone-receptor complexes. Make the other half of the complex inactive, and for K, one or more isotopes with NMR activity can be studied for its structure. The mammalian cell line nutrient medium of the present invention contains a hydroxyisotope-labeled amino acid derived from a microorganism. Nutrient media for mammalian and insect cells are known. At the same time, I also understand the requirements for cell growth, so synthetic culture-16- (Please read the notes on the back before filling in this page) Order φφ This paper size applies to Chinese National Standard (CNS) / Α4 (210 × 297 mm) 83.3 . 10,000 Printed by the Consumer Standards Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs. Yes. Some of the above-mentioned culture mediums * contain a small amount of pyrograpeic acid. If such a culture medium is required, the pyrograpeic acid added is preferably in an appropriately labeled state. Isotope-labeled pyrograpeic acid is available. Can also be purchased In the present invention, a serum-free medium is best used. In the present invention, it is best to use a serum-free medium. * A relatively pure labeled protein can be obtained by K for N MR analysis. The labeled protein is obtained from a mammal or a serum-free mammal. The ancient method of purification in cell culture media * can use any known technique. See Deutscher, HP, Guide t. Ο Ρ r ο t ft ί η Purfications. Η θ thnds in Rnzvmo 丨 〇gv. V ο I. 18 2 (19 9 0) ° The protein is fully labeled, which can be used by adding all the labeled amino acids and any other substrates used for protein synthesis. As used herein, protein synthesis includes brewing protein Biosynthesis of carbohydrate branches. The present invention provides a simple method for obtaining amino acid mixtures. This method is simple and easy to do, harmless to the user, and can be scaled up, especially by adding a large amount of labeled medium K to obtain a sufficient amount of protein for HMR The analysis is even more important. The method of generating a labeled amino acid mixture is based on a group of amino acids that have both positive and negative charges. This can be neutral compounds, or only Compounds with a positive thunder charge or only a negative charge can be isolated, which can be extracted by adsorption, or extracted from acidic and basic ion exchange resins. The proteins referred to herein are "substantially marked" or "substantially all" "The atom of a particular element in the molecule is an isotope state, and the molecule has been labeled with a sufficient amount of isotopes, thereby obtaining meaningful MMR spectral data. For isotopes with NMR activity In general, if the degree of 1SN is sufficient, its three-dimensional structure can be deduced from the NMR spectral data. Generally speaking, the given paper size is applicable to the Chinese national standard (CNS LA4 specification (210X297 mm) 83 . 3. 10,000 (Please read the notes on the back before filling out this page)

Produced by the staff of the Central Bureau of Standards of the Ministry of Economic Affairs of the People's Republic of China. Α7 Β7 V. 95% of the elements given in the invention description (M) can be transferred to the required isotope state, and the better is more than 98%. For only 2%, a sufficient amount indicates that the labeled molecule does not generate sufficient NMR interference and analyze the NMR signal of the associated molecule. Usually, it is in a sufficient amount above about 70%, and preferably above about 95%. In addition, the sufficient amount of 2Η also includes strengthening / reinforcing the nucleus with NMR activity, such as 1Η, 1 3 C, and 1 s Ν. Generally, this level of sufficiency ranges from about 20% to about 100%. The amino acid mixture starting material is a proteolysate labeled with the appropriate isotope. According to the invention, the starting protein is essentially M13C, or 13C and 15H, or 13C, 15N and at least a portion of 2H, or even only 2H. A variety of techniques have been described to produce this protein, including growing bacteria in labeled carbohydrates and salts (Kay, et a1., AjLEXsJ, growing bacteria in hydroponics of pampas (Chubb, RT, et al · ,

Biochemistry. 30. 7718 (1991)) * Growth of yeasts in algae hydrolysates (Powers, R., et ai., Biochemistry, 2J_, 43 34 (1992)), bacteria and yeasts in labeled methanol Growth (see ------ Moat, AG5, Foster, J.W., Microbial Physiology. 2d Ed., John Wiley & Sons, Hew York (1988), p. 218), and blindness Algae grow in 13C02 and / or 15N salts labeled with isotopes (CοX, J., et a 1., $, .Tg.bie—_Isotopes in P ft diatri η N ntr ί t. I η na I and Metabolic Research.

Chapman, T.E. et a 1., Eds., Intercept Ltd.,

Andover House, England (1950), p. 165). Similarly, a variety of methods for hydrolyzing proteins are known, including K hydrochloric acid, methanesulfonic acid-18-this paper size applies the Chinese National Standard (CNS), 4 specifications (21 × 297 mm) 83. 3. 10,000 ( (Please read the notes on the back before filling out this page). • Μ: *.

------ 1Τ ----— I Staff and Consumer Cooperatives of the Central Bureau of Standards, Ministry of Economic Affairs, Yinfan Α7 Β7 V. Description of Invention (frl) (LeMaster et a 1., Sm R.r a) and enzyme hydrolysis. If the enzyme hydrolysis method is used, the hydrolysis solution is easily acidified. It can denature enzymes and precipitate enzymes * and remove them from the hydrolysate by centrifugation. In the present invention, acid hydrolysis is preferred. It is preferred to use a strong inorganic acid for acid hydrolysis, such as hydrochloric acid, nitric acid or sulfuric acid or a sulfonic acid, such as p-toluenesulfonic acid or methanesulfonic acid, but it is better to use the latter. The acid concentration varies depending on the nature of the protein, but is generally sufficient for complete hydrolysis. Generally speaking, the acid concentration is from about 1 N to about 6 N, and the pH is from about 2 N to about 4 N. Acid hydrolysis is performed under non-oxidizing conditions. It can be carried out under vacuum, or by adding an inert gas such as nitrogen, ammonia, and the like. The concentration of the to-be-hydrolyzed egg Θ is about 0.5 g / 10 ml to 5 g / 10 ml, preferably about lg / 10 ml to about 2.3 g / 10 ml. The temperature and time of the hydrolysis reaction should be sufficient to cause substantially effective complete hydrolysis, but it is necessary to reduce the racemic reaction or the loss of unstable amino acids. The temperature range of the hydrolysis reaction is generally about 90 to 140 t, but in order to reduce the racemic reaction of the amino acid, it is preferably 100 to 11 5 t: the most preferable. The hydrolysis time may range from 24 to 72 hours depending on the protein 5 to be hydrolyzed. The preferred one-to-hydrolysis time is 48 hours. Amino acids that are easily cleaved by oxidation are more protected with kanogens. Preferably, a strong rhenium agent is used * which contains a sulfhydryl group * such as * ethanethiol (Fasman, G. I)., Ed., Practical—Handbook Df Bioohftmistry a n..d. Eon I 9 r BJl 〇_q ay, CRC, New York (19 8 9), p. 10). The purpose of the reducing agent is not only to protect tryptophan and weaving acids, which are easily oxidized and cracked. If thioglycolic acid * is used, it can be easily removed according to the method of the present invention. -19-(Please read the notes on the back before filling this page)

This paper size applies the Chinese National Standard (CNS). _Λ4 size (210X297 mm) 83. 3. The concentration of 10,000 A7 B7 is sufficient for ethyl mercaptan fermentation on the sprouts &gt; It ranges from about 3 to about 5¾: V / V cation exchange resin column then> Any acidic resin Simple acidic resin 9 such as the cation exchange tree Do W Chemical Co. * Midi resin ♦ Use of acidic solution in principle &gt; Any inorganic acid can be used &gt; Hydrochloric acid pR a value of amino acid> Its pH value is from about 1 to about remove all acidity and --- generally enough is 0 tube to remove the acid solution 0 for its use 2-6X column volume » Alkaline extraction of alkaline substances 〇 In principle f simple lye 9 such as 5 hydrogen is HR iR 2R 3 &gt; where alkenyl 〇 This nitrogen-containing base includes etc. lye neutralizes acidic amino acids and Basic compound i and its carboxyl group with negative lightning Printed by the Consumer Cooperative (please read the precautions on the back before filling this page) 5. Description of the invention (the use of rhenium agent to prevent damage. 7¾ ν / ν, it is better to add the hydrolysis solution to the acid. It is convenient and convenient to use. It can be used in this method. It can be 50X8-400 in this method. It can be added with hydrolysate and acidic pollutants. It is better to use simple pH value lower than most of the spin reaction. The volume of the general solution needs to be sufficient M 2 -6 × column volume is washed with water to remove the resin. * Preferred, -...-- Washing to remove acid adsorbed on the resin, but it is better to use or nitrogen-containing alkali, which is either C1-C4 or basic. The concentration of the amino group of the adsorbed acid, the neutral protected tryptophan acid and the serine acid, is between about 1 and about 0 when the ethylamine aqueous solution is drawn out. Both cation exchange resins can be used * but, for Η ♦, pyridyl * Methyl saddlery lipids include "Dowex and, M. Chigan, commercially available liquid-washing resins that can remove a neutral acid, but, for convenience, sulfuric acid, etc. Acidic solutions, however, are not low enough to trigger consumption, preferably about 2." Acid pollution. Usually M about to ensure that all pollutants are Substances * get amino acids and ketones. Use any kind of mash solution. Sodium oxide, potassium hydroxide * f, R2 and R3 are hydrogen, hydration, ammonia water, methylamine water-soluble cation exchange resin, the same. The pH value of the lye makes the amine charge, and the pH of the lye is better -2 0 _ 83. 3. 10,000 This paper size applies the Chinese National Standard (CNS) &gt; 4 specifications (210X297 mm) Central Standard of the Ministry of Economic Affairs 扃Employees' Cooperatives printed A7 B7. 5. Description of invention (<y) is higher than 10. In order to avoid the racemic reaction of amino acids under strong base conditions, its pH value should be lower than 13 and preferably between about 10-11. The test solution neutralized the acidic cation exchange resin, and simultaneously extracted the adsorbed amino acid and basic compound. The amino acid can be further purified by the anion exchange resin. The eluent obtained from the cation exchange resin was added to a basic anion exchange resin. In principle, any basic resin can be used. Preferably, a purely basic state such as hydroxide is used. Suitable anion exchange resins include Dowex 1X8-100, which is commercially available from Dow Chemical Company, and, Michigan. Amino acids can be adsorbed to aging ion exchange resins because the carboxyl group is negatively charged when the amine group is not positively charged. K lye cleaning removes alkaline and neutral pollutants. The lye used in this step may be any of the lye extracted from the amino acid in the cation exchange resin column described above. K water was used to flush the anion exchange column to remove the test solution. Preferably, no infusion residue is in contact with the amino acid bound to the resin. Therefore, it is better to wash the column with about 2-8X column volume and at least 4X volume of water. -------- The amino acid is extracted from the basic anion exchange resin with an acid. In principle * Any acid solution can be used, but, preferably, a weak acid that is easily removed and a volatile acid is used. Most preferred is formic acid or acetic acid. The concentration of the acid to be used has a value of about 2 to 6 and preferably about 3 to 5. The purified amino acid extracted from the column was a pale white solution. Another advantage of the present invention is that aspartic acid is the last amino acid to be extracted. For example, with the exception of aspartic acid, all amino acids I; 0.253; v / v acetic acid aqueous solution was extracted. Aspartic acid was 2.5 S; v / v acetic acid aqueous solution was drawn down. The present invention can therefore be used as -21-This paper size is applicable to Chinese standards (CNS (210X297 mm) ~~ '/ (Please read the note on the back before filling out this page) Printed by the Consumer Standards Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs Preparation of A7,, B7_______ 5. Description of the invention (&gt; 0) is a simple method for purifying aspartic acid. As described below, this aspartic acid can be labeled and then converted to labeled asparagine, which can be added to It is indicated in the amino acid mixture. The preferred 'the above-mentioned stripping step is a so-called "gradual gradient" stripping. At the same time, the preferred concentration of the stripping acid used is a straight line, or in addition, it is an exponential gradient method. According to the gradient used, amino acids can be gradually extracted, regardless of the M mixture or a single amino acid method. The present invention is a simple step for purifying amino acids ^ ^ simple or mixed type. The present invention is published at the same time The isolated amino acid can change the amino acid distribution in the entire amino acid mixture. The present invention indicates that * all amino acid mixtures can meet the needs of the given cell line. The isolated amino acids can be re-standardized technology, It is separated under reduced pressure or freeze-dried. Another aspect of the present invention is that the amino acid to be separated will be substantially pure, which is because the extracted acid is highly volatile. Another advantage of the present invention is related to Arginic acid is more alkaline than other amino acids. Therefore, in Yuyangli's exchange resin, sperm limb acid is the last one to be extracted, even later than all pollutants. When the pH value of the extraction When •-i〇-12, arginine is electrically neutral. Because of its overbased guanidino branch chain, when the rH value is in the range of 10-12, it will have a positive charge and neutralize the carboxyl group. Negative lightning charge. Unlike other amino acids, arginine will only be extracted from the cation exchange resin after contamination. Therefore, it can be re-isolated and crystallized. For example, the standard method of M reacts with hydrochloric acid (Cox , GJ, .1. R i η 1. Γ. H ft m ..: L8_, 475 (1928)). Another aspect of the present invention is therefore a simple purification method of arginine. Preferably, the present invention is In the hydrolysis step, a sulfur-containing hydrogen-based reducing agent is used to make the -22-I paper size applicable to the Chinese National Standard (CNS). _4 size (210X297 mm) 83. 3. 10,000 (Please read the notes on the back before filling this page) Order 1 &gt; A7 __ ^ ____ B7 __ 5. Description of the invention (Pure amino acid mixture maintains the same ratio as the initial hydrolysate, only Very small changes in yield. Another aspect of the present invention is a simple method for preparing a pure amino acid mixture to maintain the same ratio as the starting protein. Therefore, the ratio of the amino acid can be selected by selecting a suitable The starting protein or protein mixture is controlled. In addition, the amino acid mixture 'prepared according to the present invention may be further added with commercially available or synthesized amino acids or acids. For example, in mammalian cell culture medium, cysteine is an extremely important amino acid, but its concentration in most bacteria, yeasts or thin proteins is not sufficient to feed mammalian or insect cells. Animal protein biosynthesis. _ Known enzyme methods for synthesizing cysteine. For example, it is listed here as a reference material]]. [.8. Patent 4,733,011 to ^ 73113 "mentioned in 3 et al. Using hydrogen sulfide 'M enzyme reaction to produce L-serine into L-cysteine The method is listed here in the reference 13 卩 丨 ^ 1: 3 et al. 1]. $. Patent 4,782,021 mentions that K serine hydroxymethyl convertase reacts lysine with formaldehyde to produce L -Serine. Isotope-labeled cysteine, including i3C-cysteine and 15'-cysteine, its preparation uses commercially available substrates to participate in the steps-* --- Central Bureau of Standards, Ministry of Economic Affairs Employee Consumer Cooperatives printed the above-mentioned method using enzymes to prepare isotope-labeled cysteine is not conducive to the production of triple-labeled 2H, 13C * 15N -cysteine, because it is not possible to obtain a proper triple-labeled starting material. Even if it is available This substrate, dehydrogenation, adversely affects the kinetic reaction of the enzyme. According to this, the present invention also provides a novel 'method for the isotope labeling of cysteine with M, including the triple labeling of cysteine, and including this A mixture of amino acids of cysteine. The isotope indicates cysteine Method, including triple labeling of cysteamine-23-83. 3. 10,000 (Please read the cautions on the back before filling out this page) This paper size applies to Chinese national standards (CNS LA4 specification (210 × 297 mm) I) I) Printed by the Consumer Standards Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs A7 __ · ___ B7 V. Description of the invention (j) The pure amino acid mixture maintains the same ratio as the starting hydrolysate, with only a small change in yield. Another aspect of the invention is a simple method for preparing a pure amino acid mixture to maintain the same ratio as the starting protein. Therefore, the ratio of the amino acid can be controlled by selecting an appropriate starting protein or protein mixture. In addition, the amino acid mixture * prepared according to the present invention may be further added with commercially available or synthesized amino acids or acids. For example, for mammalian cell culture media, * cysteic acid is an extremely important amino acid, However, in most bacteria, yeast, or algae proteins, the concentration is not enough to provide mammalian protein biosynthesis to mammalian or insect cells. B. The enzyme method for synthesizing cysteine. Example For example, it is listed here for reference: ^ US Patent 4,733,01 1 to Miyahara et al. Mentions a method for preparing L-serine to L-cysteine by hydrogen sulfide 'W enzyme reaction. Listed here Ishiwata et al.'S IKS. Patent Reference 4,782,021 refers to M. Serine hydroxymethyl converting enzyme reacts lysine with formaldehyde to produce 1 / $ _ acid. Cysteines are labeled by isotopes, including 1SC_cysteine and 13C * Cysteine, which is prepared using commercially available substrates. The use of enzyme methods to prepare isotope-labeled cysteine is not conducive to the production of triple-labeled 2H, 13C, UK. -Hemicarbic acid, which is unable to obtain a proper weight labeling starting material. Even if this substrate is available, dehydrogenation has an adverse effect on the kinetic reaction of the enzyme. Based on this, the present invention also provides a novel method 'Isotope-labeled cysteine, including triple-labeled cysteine' and an amino acid mixture comprising the isotope-labeled cysteine. Method for W isotope labeling of cysteine, including triple labeling of cysteamine_-23- gen. U4 specification in paper size (21〇 &gt; &lt; 297mm) '83.3. 10,000 (Please read the back first (Note ^ then fill out this page)

Ministry of Economy _ Printed by A7 B7, Employees' Cooperatives of the Bureau of Standards and Labor. V. Description of the invention (Acid, hydrolyzes the protein M containing the labeled hemi limb amino acid. Cysteine is known to be unstable under acid hydrolysis conditions Therefore, the method includes, n protecting cysteine during proteolysis' ί) separating the protected hemicanine and iii) deprotecting and isolating the labeled hemicarine. K S-benzyl ether is known to chemically protect thiol groups. (See Gre e π, T. and Wats, P-G.M., Pro'hfini'.i vp fi γ o u d Chemistry, 2nd Ed. * John Wiley &amp; Sons, Hew York (1991)). Fact i: In the chemical synthesis of peptides, that is, KS-benzyl-protected cysteine. However, this protective compound is not stable in acid, and at the same time, it will be broken under the condition of hydrolyzing protein. It is known that when the thiol group M is protected as a charged benzyl thiolase in the following formula, it is stable under the conditions of strong hydrolyzable protein and hot acid conditions *

'⑴-where Ri. Is cysteine, either alone or part of a protein molecule * and at least one of R2-Re is an acidic group, such as a carboxyl * C2.-C4. Carboxyalkyl or Cn -C ^ carboxyalkoxy or a base such as dialkylamino, C3-Cedialkylamino * alkyl or C3-C0dialkylhelylalkoxy, and the remaining R2-Rs, regardless of Is alone or in combination * is hydrogen, halogen, Ci-Ce alkyl or Ci-Ce alkoxy. Preferably * R «is an acid group such as carboxy, carboxymethyl or carboxymethoxy, or a base such as dimethylamino * dimethylaminomethyl or dimethylaminomethoxy * and its- 24-This paper size applies to Chinese national standard (CNS 丨 / 4 size (210X297mm) 83. 3. 10,000 (Please read the precautions on the back before filling this page) Clothing — nn TJ n ^ i —ϋ I— II m 11 · i- 0 ij i 5. Description of the invention (&gt; A7 B7

The remaining R 2-R., either alone or in combination, are hydrogen, dentine, C: L-CS alkyl or "-Ce alkoxy. Telluryl cysteate (II) is halogenated Reaction of benzyl compound (III) can produce compound (I) XR ^ H (II) in high yield

I— \ II (Please read the precautions on the back before filling out this page) Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs, where X is the stability of halogen in alkaline solution in (I). Its analogue uses known HD * eg in acetic acid. In the present invention »methyl thioether, the reaction is preferred, and the glass is charged, so the" protective molecular method, and its technology, can maintain the preferred (IV) state in the alkali metal as chlorine or bromine, and as defined above , Such as diluted ammonia, or aqueous sodium hydroxide solution. The charged acid or base can strengthen the benzyl sulfide in the acid solution. The starting thiol is protected under hot acidic conditions. ’It can be separated by other solubility, ion exchange resin and other molecular species. Convert the alcohol derivative (I) into the starting thiol and react with it * For example, dissolve sodium in liquid oxygen or sodium, cysteine UII) with acid p-carboxyl group, V〇-RI R'- HN-CHI H, CS-

, 〇H, νβ '(IV) 25 This paper size applies to Chinese National Standard (CNS) .. A4 size (21 OX 297 mm) 83.3. 10,000 Printed by A7,, B7 of the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs Description of the invention (0. When the reaction starting material is a free state cysteine *, then R and R 'are gas' or when the reaction starting material is a polypeptide, R and R' represent the middle phase of the polypeptide O Amino acid. 'Reaction of cysteine (11), whether in free form or in the peptide unit of the peptide, with p-chloromethylbenzoic acid under mild alkaline conditions' For example, in diluted Caishui or sodium hydroxide aqueous solution, high yield of compound (IV) can be prepared. Isotope-labeled cysteine * is prepared. An appropriate isotope-labeled protein is produced under the conditions of thioether formation in diluted ammonia. Or sodium hydroxide in aqueous solution to react with (111). Known suitable proteins can be used, but it is preferred to use egg prion derived from purple sulfur bacteria, such as Rhodopseudomonas and oaosu lata, and other cysteine-rich organisms , Such as Leptot. Hr ΐ y discophora and Schizophvl lum commu ne »and recombinant bacteria that use K to produce cysteine-rich proteins, such as ATCC 31.448, and E. coli expressing human insulin / \ _, can be used to grow the organism in suitable labeled media. Cysteine derivatives (IV) are stable in hot, strong acid medium, but are less soluble in acid and neutral medium at room temperature. Therefore, the compound --- XIV) can easily interact with protein hydrolysates Other amino acids are separated, for example, hydrolyzed or centrifuged. In contrast, cysteine derivatives (IV) have high solubility in rotatable organic solvents, such as ethanol hinges. Therefore, extraction with alkaline organic solvents, such as aqueous ethanol ammonium compounds (丨 V), can be easily hydrolyzed with proteins The other components of the product are separated. The 'cysteine derivative (IV) can be isolated from the protein hydrolysate by a modification of the aforementioned amino acid purification method. The protein hydrolysate was adsorbed on the ion exchange resin in H + state, and the M acid was extracted to remove the pollutants-Λ __-26-This paper is applicable to the standard (CNS) / 4 (21〇297297 mm) &quot; ~ ~~ ^ 3 (Please read the notes on the back before filling out this page) Order the consumer cooperation of the Central Bureau of Standards of the Ministry of Economy Du printed A7 _; _____ B7 V. Description of Invention (yT). After washing with water, the amino acid of the hydrolysate, including the cysteine derivative (IV), was extracted from the 1T resin, added to the 0H-resin, and extracted with ammonia. After washing with water, the diluted acetic acid stripped the amino acid from the 0H-resin. However * because the benzoic acid branch of the cysteine derivative (IV) * makes it more acidic than other amino acids, the compound (IV) is extracted after all amino acids, including aspartic acid. Isolation of the cysteine derivative (IV) is carried out by drying or concentrating the appropriate liquid separation, and then concentrating the resin, and then diluting it with diluted ammonia water, and then allowing the appropriate liquid separation to evaporate. It can be easily converted back to cysteine (II) by standard methods. The cysteine derivative (IV) can be easily converted to cysteine (II). It is preferred that the alkali metal be dissolved in a suitable solvent for reaction, for example, liquid ammonia or ethanol. The admiration. Cysteine can be generated by the above methods without labeling, single labeling, double labeling or triple labeling. The required isotopic composition of the cysteine-containing protein starting material can be controlled by controlling the composition of the nutrient medium used to produce the protein. It is known that mammalian cell culture medium, in addition to amino acids and glucose, still contains a variety of compounds, such as vitamins, fatty acids, must be minerals and growth --- toned? Another aspect of the present invention is that the purely labeled amino acid mixture produced by the present invention can be added to the growth factor mixture required by any cell line, thereby producing a mammalian or insect cell line. Isotopically labeled media. In addition to isotopically labeled amino acids, all other substrates can be used by cells for protein synthesis. For example, a carbohydrate * such as glucose may be in the K13c-labeled state and / or in the hydrogen state. At the same time, it is known that the proteolytic step will destroy the amino aspartame and bran (please read the precautions on the back of Jing before filling this page)-'' © --- This paper size applies to Chinese National Standards (CNS). _Λ4 specifications (210X297 mm) 83. 3.10,000

«I« I Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (/) Amidoamine, which is accompanied by the production of acidic amino acids, aspartic acid and glutamic acid. Most mammalian media contain large amounts of glutamine. However, for some mammalian cells * the addition of glutamine to the amino acid mixture produced by the present invention does not affect its growth rate or the productivity of the recombinant protein. However, for some mammalian or insect cell lines, the addition of glutamine is extremely important for its growth performance. Another aspect of the present invention is that, according to the characteristics of the cell line used in the culture medium, it is not necessary to add the glutamine to the amino acid mixture produced by the present invention. It was also found that no racemic reaction was found using the better conditions described in this section. It is known that the glutamic acid and amino acid mixture can be easily separated by using a chromatography method, and then converted to glutamine by a κ chemical or enzyme method. The resulting glutamine is then used to add to the desired amino acid mixture. When the pH is neutral, only the glutamic acid is negatively charged in all amino acid mixtures, because it has two carboxyl groups and only one amino group. The remaining amine-based acids can be uncharged (e.g. glycine) or positively charged (e, g. Lysine) at neutral pH. Therefore * after removing the acid extraction solution, eg Μ freeze-drying, the amino acid mixture is passed through an anion exchange resin prepared by the weak acid, and the glutamic acid and the amino acid mixture can be specifically separated . In principle, any weak acid can be used, but preferably, an anion exchange resin of acetate or formate is used. When other neutral or positively charged amino acids pass through the anion exchange resin, the glutamic acid can be adsorbed on the resin because it has a negative charge. After the amino acid is washed out from the resin, the resin is washed with M water, and the glutamic acid is washed out from the column with an appropriate acid solution. Standard methods can be used, such as decompression evaporation or freezing-28-This paper size applies Chinese National Standard (CNS). &Gt; 4 specifications (210X297 mm) 83. 3. 10,000 (Please read the precautions on the back before filling in this Page) .7 "Packing ·-* A7 B7 Five invention description (drying method, recovery of the separated glutamic acid. The known method, such as the enzyme method published by Fes ί keta 1, can be used to convert the bran Uric acid is converted to glutamine. However, the enzymatic method as described above is not conducive to deuteration and triple labeling. In addition, according to the present invention, a novel chemical method of the following scheme 1 can be used. Flow stalk 1

Ho. Y0

X-HN-CH

\? XN-CH XN-CH CH, 1 ch2 1 ch2 1 ch2 I ch2 1 ch2 I 1. -CHI CH, (Please read the notes on the back before filling out this page) Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs, h2n, 0 (IX) H. , 〆I HjN -CHI ch2 ch2I c H2N〆 ,, 0 (X) -29-This paper size applies to China National Standard (CNS) __ A4 specification (2IOX297 mm) 83. 3. 10,000

»I A7 _ _ B7 V. Description of the invention ρβ). First, the amino group of glutamic acid is protected to obtain (VI). Many suitable protecting groups are known, including .Fmoc, t-Boc and the like. N-protected glutamic acid is cyclized to form oxazolidinone (V 11). This conversion method is known 'e.g. See also, Itoh (Cham. Ph ^ rm. Rull, _ 11_, 1 (3 7 9 ((19 6 9))). In the 11 oh method, the catalytic amount In the presence of a strong acid such as benzylsulfonic acid, the compound (VI) is reacted with paraformaldehyde to obtain (VII), which can be separated by known methods. The free carboxylic acid of (VII) is activated with a suitable activator Groups, such as dicyclohexylcarbodiimide, diisopropylcarbodiimide, η-butadiene bromide limbs and the like. At the same time, other activating groups are known. Activated oxazolidine _Reaction with ammonia gas to obtain the cyclic amidine derivative (VIII). When preparing and labeling glutamine, ammonia gas must also be labeled with 15N. Like all materials, ΐ5ΐ-ammonia is very expensive. Even more * It is a gas * so it is difficult to be accurately controlled and measured. Therefore, it is preferred that 15 N-ammonia gas is generated in another flask by adding a 15 N salt solution, such as 15N-ammonium chloride, Ammonium sulfate, etc., together with human lye to react with it, the generated ammonia gas is reacted with activated oxazolidone. Suitable solvents --- --- the agent includes polarity, hydrogenation Organic solvents, such as those printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs of dimethyl chloroform and dimethyl formamide (please read the precautions on the back before filling out this page) hydrazine, and suitable lyes include, strong or The strong mutual soluble alkali solution of hydrazone, such as tetramethylguanidine and sodium hydride. In a suitable solvent, the cyclopentamine derivative (V 11 I) Μ weak alkaline aqueous solution is hydrolyzed to obtain H- Amine derivatives (IX). Various lyes can be used * However, for simplicity, a simple lye, such as sodium hydroxide dissolved in an aqueous ethanol solution, is used. In order to reduce the hydrolysis of amidine, it is preferred to use only 1- 5 When the most lye is better, use 1-2 equivalents. __- 30 -__. This paper size is applicable to China National Standard (CNS) Xi 4 specifications (210X297 mm) 83.3. 10,000 A7 B7 V. Invention Note ($) Use of glutamine for deprotection depending on the protecting group used. (See Green, Fermented amine (X) e · g. M Crystallization method or Chromatographic method known under reduced pressure. Known method of loading It can also be used for the above-mentioned hydrolysis conditions of another aspect of the invention, and the preparation of asparagus The method of amidine can prepare the labeled amino acid mixture. The method of the present invention can purify the amino acid mixture labeled by any isotope only about the amino group and carboxyl group of the amino acid. Labeled, all mammalian or insect cells grow. The mammalian or insect cells and their isotope mixtures are completely labeled. The present invention will be described by the following examples. N-protected eta 1. f supra). It can be separated, evaporated, or purified, and e · g · K aspartic acid can be converted to asparagine. Cysteine, glutamate M 2 Η, 1 3 C and 1 s Η are labeled as any group. Another aspect of the invention is the use of substituted base acids of the present invention. Since the sun and the moon are hydrogenated, any of them can be purified by applying the present invention together. The obtained amino acid mixture is sufficient to supply. Therefore, the other aspect of the present invention is a metabolite, which will be the same as the starting material. It will only be described, which does not limit the example of the development. Preparation Example The algae biomass obtained from ChTnrft1a medium was diluted to about 10% sticky bark by M (water), and then placed on ice. Όί Twilight 0 spoon viscous microliquefier (Microfluidizer) three times to make cells rupture. Set W, one, and thick solution K 5, 0 0 0 r p m, 5 t: Centrifuge for 15 minutes. Collect the supernatant 柩 wr U &gt; half a step twice. The precipitate was dissolved in water and centrifuged under the same conditions.恵 Repeat this step 31 83. 3. 1〇. ° 00 (Please read the precautions on the back before filling this page)

This paper size applies the Chinese National Standard (CNS). &Gt; 4 specifications (210X297 mm) printed by the Consumer Cooperatives of the Central Bureau of Standards, Ministry of Economic Affairs, A7, __B7___ 5. Description of the invention (Mix all the supernatants and add trichloroacetic acid (Final concentration 5 3: v / v), store overnight at 5 ° C; store the resulting suspension at 5,000 rpm * 5 t: centrifuge for 30 minutes. Pour off the supernatant, and dissolve an equal volume of propylene. After sinking, it was centrifuged for 15 minutes at 5,000 ppm * 5 C. The supernatant was removed, the precipitate was dissolved in 500 ml of ethanol / ether * and collected under reduced pressure. The precipitate was washed with ethanol / ether And then dried under reduced pressure. — Separate the above 7 g of soluble protein * in Zhenyi with 3 M methanesulfonic acid (70 m 1) containing 4% v / v hydrothioacetate at 100 t The reaction was allowed to proceed for 48 hours. After cooling, the obtained hydrolysate was slowly poured into water (70 m 1), then cooled in an ice bath and the resulting mixture was allowed to stand for about 10 minutes. The obtained cooling solution was centrifuged ( RC-3B, 250 ml centrifuge tube, 5,000 rpm, 10 minutes). The supernatant was injected (36m! / Min), and FMI laboratory QSY (FMI Lab Pump QSY) column of Dovex 50 X 8 ion exchange resin (H + state, 500g). After all the hydrolysate has been injected into the column * then the diluted telluric acid aqueous solution (PH2.0 , 1.5L) and water (2L) (3 (3ml / min) into the column -----, collect and separate the liquid from the bottom of the column (labeled H *, 500ml). Use a pump to connect the H + column with the D〇Wex 1X8-100 ion exchange resin column is connected (0H-state * 500g). The diluted ammonia water (1% v / v, 9L) is driven into the pump (36nil / .min) by the H * column with a pump, and The eluent was allowed to flow into a 0H-column. The liquid was collected from the bottom of the OH_column (labeled oh- * 500ml). Analyzed by TLC (Analtech Silica GS plates, n-BuOH:

AcOH: Ha 0 (2: 1: 1 v / v) developing solution: Ninhydrin (1 χ v / v in MeOH: Glac AcOH (97: 3 v / v)) shows spermine at a- 32-This paper size applies to China National Standards (CNS) &gt; 4 specifications (210x297 mm) 83. 3. 10,000 (Please read the precautions on the back before filling this page) •-^ 衣 ------ Order ------- β-- ill----· Consumption cooperation between employees of the Central Bureau of Standards, Ministry of Economic Affairs, printed A7 ______B7__ V. Description of the invention 〇l) 0H-13-18 appeared in the liquid separation. Wait for the arginine solution to come out, stop the pump, and connect it directly to the 0H-column. When the separation and collection by the 0H-column (500) was started again, the pump was restarted. When the 500rol separation was continuously collected from the bottom of the column, the diluted acetic acid aqueous solution (0.25% v / v, 1 〇l) Hit by a 0H-column. The performance lotus pump was used until the Ningdling reaction no longer appeared in the eluent. TLC analysis (Analtech Silica GS plates, n-BuOH: Ac0H: H20 (2: l: lv / v) developing solution: Ning box delin (1¾ v / v in MeObGladcOH (97: 3 v / v)) shows amino acid mixture (in liquid OH-25-36) ° will contain spermine The acid separation and amino acid mixture were separated for freeze-drying for drying and concentration. After the solution was dissolved in water, it was filtered through a 0.22 Wm filter membrane into a sterile bottle, and then freeze-dried. Basically, according to C 〇x,., AjiP.ra steps, the arginine acid was separated and crystallized to produce hydrochloride to obtain 0.31 g. The amino acid mixture was separated and separated to obtain a white-yellow powder, 4.6 g 〇 ——- -Example 2 Obtain serum-free CH0-SSFM-1 medium (Gibco) from the supplier * It is suitable for CH0 cells, and B removes the amino acids from it. Prepare as follows: Add 200 ml of CH0-SSFM-1 aliquots without amino acid, and add 1. A mixed amino acid (3 4011 ^) + cysteine (2〇1 ^ ) + Crystalline amino acid (4〇1 ^) 2. Mixed amino acid-33-This paper size applies Chinese National Standard (CNS) &gt; 4 specification (210X297 mm) 83. 3.! 0, 〇 〇〇l · --1--; --- ^, J clothing ------, 玎 ------ ο (Please read the precautions on the back before filling this page) A7., B7_ ' _ 'V. Description of the invention (5V) (340mg) + Cysteine acid (20i «g) + Crystalline amino acid (40mg) + Bonded amine (I20mg). Use M 0.22 w 2 Membrane filtration made this solution sterile, and the aliquots (2-3ml) were seeded into CHO cells (initial concentration lx105 / rol). The cell number and% were recorded relative to the control CH0-SSFM-1 medium sample Viable cells (%): 48h_Z2h_96h_120h_144h 168h Control group 2.4 (99) 5 · 2 (96 &gt; 々.0 (93) 7.0 (91) 5.6 (83) 4.0 (64) 1 1.6 (99) 3.8 (93) 5.1 ( 97) 12.0 (92) 8.2 (84) 5.0 (72) 2 2.2 (99) 4.0 (95) 5.5 (93) 7.2 (95) 7.2 (82) 7.0 (69) The results show) amines obtained by the example method Base acid mixture 'its growth characteristics and control The control group is not different and 1) the addition of glutamine is not necessary for cell growth. Example 3: A double-labeled amino acid was prepared essentially according to the method of Example 1, except that the algae biomass was taken from rb 1 or ft 1 a as the sole carbon source and nitrogen source, respectively M 13 Coz and K15N〇3. Medium. 13C, 15N-cysteine is prepared according to the above-mentioned enzyme method. Printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs. Obtain CH0-SSFM-1 medium (G ibco) from the supplier, which removes amino acids, carbohydrates, intermediate metabolites and protein hydrolysates. To the above 1 liter of medium: 13C, 15N-mixed amino acids (including 13C, 15N-streptane acid): 3 g of 13C, 15tr-arginine-HC1: 240 mg ί% * Cysteine Amino acid: 160 mg -34-83. 3. 10,000 (Please read the notes on the back before filling out this page) This paper size applies to China National Standard (CNS) / A4 specifications, (210X 297 mm) Central Standard of the Ministry of Economic Affairs A7,, B7 _____ printed by the Bureau's Consumer Cooperatives V. Description of the invention (3¾ 13C -Sodium pyruvate (Isotec + Inc.): 70 mg 13C-D-glucose: 3. 8 g K 0. 22 μ Sterilize by sterilization. CH0 cells are added to the above solution at 250ml, and they are expressed by K and reconstituted with human chorionic gonadotropin (hCG) to a final concentration of 3 × 10e cells / ml. After stirring this culture solution for 15 hrs The cells were separated by centrifugation. The cell pellet was dissolved in 500 Hi 1 as prepared above. 1 3 C, 1 s N-labeled culture medium to a concentration of 1.2 X ...., ν, 10 β cells / ml And stirred for 7 3 hours. According to the same limitation, a commercially available CH0-SSFM-1 medium was used as the control group. Anti-hCG antibody was used The concentration of 13C and 15N-labeled hCG obtained by M RIA was determined. At 73 hours, the concentration of 13C and 1SN-labeled hCG was 90 pntole / ml ', which was 88 hr., 100 pmole / ro compared to the control group. The concentration of h CG is not stated. Example 4: Preparation of Cysteine-S-4 -Methylbenzoate Ammonium Cysteine (2.42 g, 20tnmol) and 4-chloromethylbenzoic acid (3.74g (20 mmol) was dissolved in water (90 ml) *, and concentrated ammonia water was reacted with it. The resulting solution was stirred at room temperature * for 1 hr. The solvent was removed by evaporation-about -50 ffl1 to obtain a precipitate. Dropwise addition Concentrate the ammonia water until the precipitate is dissolved. Then remove the solvent in the volatile mode and the precipitate will reappear. Add anhydrous anhydrous distillate to obtain the white crystals of cysteine-S-4-methylammonium benzoate (intermediate value: C, 49.0: Η, 5.8; Ν, 9 · 3; S, 11 · 6, theoretical value C, 58.5 Η, 5.9; H, 10.3; S, 11.8); yield 4.9 g (90S;), Rf (2Π: 1 ν / ν / ν n-Bu0H: H20: Ac0H) 9.73. Delta Η (D2〇, 300 Mhz) 7.76 (2H, d, J 8Hz), 7.37 (2H, d, J 8Hz), 3.78 ( 2H,

A -35-l The paper size applies the Chinese National Standard (CNS) &gt; 4 specifications (210X297 mm) glTllO ^ (Please read the precautions on the back before filling this page) Order ----------- Printed by the Consumer Standards Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs A7 B7 V. Invention Description. S), 3.73 (1H, dd, J2, 5 Hz), 2.90 (2H, m), Delta C (D20, 75 Mhz) 175.30, 172.89 , 141.18, 135.43, 129.34, 128.86, 53.60, 35.178, 31.78, 28.16. Example 5: The stability of cysteine-S-4-methylbenzoic acid to acid hydrolysis conditions was added to the Schlenk test tube. Cysteine-S-4-methylbenzoic acid ammonium (lg) was dissolved in water with frozen methanesulfonic acid (3M) and thiohydroacetic acid (4%). Use a vacuum pump to remove morale, seal the nozzle, and heat it to 100 Torr, 48 hrs. After it was allowed to cool, cysteine-S-4-methylcrate ammonium formate was separated by centrifugation, and water was washed with quantification method M (3x50π &gt; 1). iOU .: Separation of cysteine from cysteine-S-4-methylbenzoate ammonium Cysteine-S-4-methylammonium benzate (200 mg, 0.76 mmol) was added to both necks Pear-shaped bottle ... The flask was cooled in dry ice / propane and flushed with K ammonia. After about 10 m of ammonia was concentrated, nanometal (about 50 mg) was added every 1 minute until indigo appeared permanently. Add dry ice (approximately ntg) as soon as possible to produce a white precipitate. The ammonia was evaporated and the dry ice was reacted with the residue. When all ammonia has evaporated, add water (10 m 1) and carefully add concentrated

------ hydrochloric acid, adjust the pH value to about 8. The mixture was allowed to generate gas for 24 hours, and the pH was adjusted to about 5 with K concentrated hydrochloric acid, and then the suspension was stirred. The resulting precipitate was collected by filtration, dissolved in degassed water (approximately 10 ml), and cMthiothreitol (114 fflg) was added thereto, followed by stirring under nitrogen for 5 hours. The resulting suspension, M W h a t m a η 〇 〇 .Australia was filtered 'and volatilized to give a gum. Two groups of white crystalline cysteine were obtained by dropwise addition of anhydrous distilled ethanol, and the yield was 30 mg. Example 7 Isolation of cysteine from lysozyme The size of the paper is applicable to the Chinese National Standard (CNS) / 4 (2 丨 〇 &gt; &lt; 297 mm) 83.3. 10,000 (Please read the notes on the back before filling (This page)

A 7,, __ B7 __ V. Description of the invention (. Dissolve lysozyme (10 g) in water (1 L), add trichloroacetic acid (50 g), and store at 4 t overnight. Isolate the obtained by centrifugation The precipitate was washed with K acetone (300 ml, x3) and dried. The yield was 9.95 g. Under nitrogen, the denatured lysozyme was dissolved in degassed water, and then dithiothreitol (2.14 g) was added. Shake overnight at room temperature. Add anhydrous distilled acetic acid (200 ml) and centrifuge at K5k for 10 minutes. Re-dissolve the precipitate in B (200 m) and centrifuge again. Repeat the last procedure. 4-Chloromethylbenzoic acid (1.54 g) was dissolved in degassed water (42 ml) and concentrated ammonia (7 in 1). The resulting solution was added to the separated precipitate and shaken overnight at room temperature under nitrogen. Add anhydrous distilled ethanol (200 m 1), and centrifuge at 5 K for 10 minutes. Dissolve the precipitate in ethanol (200 ml) and centrifuge again. Repeat the last operation step. In a dry box, place all The separated precipitate was separated. K was washed with ethanol and found to contain a protein-like substance. So * mix the above The supernatant was centrifuged at 5 k for 10 minutes * to separate the precipitate. Add degassed water and dithiothi-eitol, and shake overnight at room temperature under nitrogen. Add acetone (10 ml), and It will be centrifuged for 10 minutes in 5k. Printed by the Consumer Cooperatives of the Central Bureau of Quasi-Staff of the Ministry of Economic Affairs (please read the precautions on the back before filling this page). Dissolve the precipitate in degassed water (125 ml) and dissolve it in Water (21 ml) of 4-chloromethylbenzoic acid (0.77 g) and concentrated ammonia (4 ml) were reacted. Then under nitrogen, shake at room temperature overnight. Add acetone (100 ml), and it will be in 5k Centrifuge for 10 minutes. After drying the obtained precipitate in a drying cabinet *, mix it with the previously obtained precipitate. Its total yield is 9.5 g (87%, according to the lysozyme amino acid sequence). -37 -This paper size is in accordance with Chinese National Standard (CNS) _A4 (210X297 mm) 83. 3. 10,000 Printed A7 by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (more than 0 will divide the obtained protein into Solution (9.5 S) in 3M methanesulfonic acid (95 ml) containing 4X v / v hydrothioacetic acid under vacuum * 100 t Heat for 48 hours. After cooling, mix the hydrolysate with water (9 5 m 1) * and pour the resulting mixture (50 ml / min) on its base. Dowex 1X2-400 equipped with FM1 laboratory pump QSY The state of the ion exchange resin column '250 g) ° When all the hydrolysates K. pump into the column' diluted (pH2, 0 1L) and water (1.25 L) into the column (50 mi / min). Using a pump, connect the H * column to Dowex 5 (^ 8-100 ion exchange resin column (01 ^ state 1250 g). Dilute ammonia (2 3: v / v, 6 L) K Pump into H + column (50 ml / min), and punch the eluent into oH_ column. Collect the liquid separation (2L) from the bottom of oH_ column. TLC analysis (A na 11 ech S ί 1 ica GS p 1 ates, η-B u 0 Η: / \ c0H: H20 (2: l: lv / v) Developing solution: Ning Xidering (1¾ v / v in MeOH: (Hac AcOH (97: 3 v / v)) shows that spermic acid appears in the liquids 2 and 3. After the spermine liquid is flushed out, stop the pump operation, and connect it to the 0H --...- tube column, K water flush (2L). When the liquid is continuously collected and separated by the 0H-column (2L) * The pump is turned on again. When the 50ml liquid is continuously collected from the bottom of the column, the pump is charged into the diluted aqueous solution of acid (0.25 ¾ v / v, 〖〇L). Continue pumping until no more Ningdrin appears in the eluent. TSTLC analysis (Analtech Si l.ica GS plates, n-BuOH: v / v) The developing solution Ning Xidering (U v / v in MeOHUac AcOH (97: 3 v / v)) showed the emergence of the amino acid mixture (-3 8 ~ this paper ruler S National Standards (21〇 &gt; &lt; 297 Publication 1 (Please read the precautions on the back before filling this page)

Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 ‘, B7 V. Description of the invention (&gt; 7). In liquid separation 11, 12 and 1 3). When 1 L of liquid was collected from the bottom of the column, the diluted acetic acid aqueous solution (2.5¾ v / v, 12 L) was pumped into the 0H-column by a pump. The pump continued to operate until Ningdrin was no longer detected in the eluent. Analysis by TLC (A na 11 ech S Π ica GS p 1 ates, η-B u 〇 Η: Ac0H: H20 (2: l: lv / v) developing solution Ning Xiedlin (uv / v in MeOH: G) ac AcOH (97: 3 v / v)) shows the presence of aspartic acid (on liquid separation 15-18) and cyste &amp; acid-S-4-methylbenzoic acid (on liquid separation 18-25). Will contain The separation phase of cysteine-S-4-methylbenzoic acid, and the pH value adjusted by 2 diluted sulfuric acid to 2. The resulting solution K25 ml / min was passed through Dowex 1X2-400 ion exchange resin (H * State, 250 g). After all the hydrolysate has been charged into the column, then the diluted sulfuric acid aqueous solution (fH2.0, 500 ml) and water (500 ml) are charged. The diluted ammonia water (2 % v / v, XXL) into a column (25 ml / inin). The liquid was recovered from the bottom of the 0H-column (approximately 15 (} 1111). TLC analysis (Anaitech silica GS plates, n-BuOH: — — —FtC〇H: H2〇 (2: l * .lv / v) developing solution Ning Xidlin (1; K v / v at

MeOH: Giac AcOH (97: 3 v / v)) showed the appearance of cysteine-S- (4-methylbenzoate) ammonium in separation 12, and the cysteamine was obtained as a white powder after volatilization. Acid-S-U-methylbenzoate) ammonium, yield 0.47 g (3296, based on lysozyme derived). Fan: Separation (50% 2H, 13C * 15M-labeled cysteine)-S-4-methylammonium benzoate

10 g of 50 g containing glutamic acid (as prepared in Example 9) 2H, 13C -39-

Applicable to China (CNS X 297 Public Director) 83. 3. 10,000 (Please read the precautions on the back before filling out this page) Clothing 丨 _ n _---II-m L- i---1 I mn ^ — ------ I— I -1 I-- Consumption cooperation by employees of the Central Bureau of Standards, Ministry of Economic Affairs, Du printed A7 B7 V. Description of the invention (M) *-The standard amino acid mixture is dissolved in 436.2 ml of water and 436.2 In ml of deuterated water (D2〇), it contains (8 g), Na (: 1 (5 * disc buffer (cH7,100 ml), TK-M metal salt (20 ml), calcium chloride ( Concentrated solution, 4 ml), vitamin mixed solution (1.4 mn, riboflavin (2 ml) * P-aminobenzoic acid (0.2 ml). The resulting solution was sterilized to make it sterile and evenly divided into 500 ml. In the Zhenlian triangle flask, plant ATCC 31 448 (―a large intestine rod that expresses human insulin a chain engineered by heavy sister engineering) into the solution, and shake in 37¾ for 24 hours. Centrifuge the culture solution and separate the The obtained cell pellet was freeze-dried to obtain about 1.1 g of dried material. Then Mdithiothleitol and 4-chloromethylbenzoic acid were reacted with the cell pellet and hydrolyzed by the method according to Example 7. Except using 2 5 g of

Except for Dowex 1X2-400 ion exchange resin (H + state) and Dowex 50X8-100 ion exchange resin (0 Η-state), the biomass obtained was purified according to the method of Example 7. The eluate contains (50% 2H, 13C, 1SN-labeled cysteine)-S-4-ammonium methylbenzoate, and the cH value is adjusted to 2 with diluted sulfuric acid. The resulting solution K10mi / min was passed through &amp; 0wex 1X2-4 00 ion exchange resin (H + state, 5 g). When all the hydrolysate has been pumped into the column, then dilute the sulfuric acid solution (2.0, 200 ml) and water (100 ml). Pour diluted ammonia (2¾ v / v, 1L) into the (10 ml / min) column. Collect and separate the liquid (about 15 ml) from the bottom of the 0H-column °° C analysis (Analtech Silica GS plates, n-BuOH:

AcOH: H2〇 (2: 1: lv / v) developing liquid Ning Xidering (1¾ v / vm MeOH: Glaq AcOH (97: 3 v / v)) showed that it appeared a sister component, which -4 0 &quot; This paper size applies to Chinese National Standards (CNS), 4 specifications (210X297 mm) 83. 3. 10,000 (Please read the notes on the back before filling out this page), -0 S. Employee Consumer Cooperatives, Central Bureau of Standards, Ministry of Economic Affairs Printed. A7,, B7 V. Description of the invention (today ^ /) "The value is the same as the cysteine-S- (4-methylbenzoic acid) ammonium prepared in Example 1. The appropriate liquid separation is evaporated to give (50 % 2H, 13C, 15N-labeled with cysteine) -S-4-methylbenzoic acid ammonium as white powder, yield 30mg .. SLJO- f: h lorel la sp culture solution in each M &gt; 98% 1 3C02 and &gt; 98% are the main carbon and nitrogen sources in 1: 1 v / v water / dehydrogen water. Dilute the resulting algal biomass (about 500 g) M (water) to about 10% ~ .V. After being viscous, place on &amp; ice, and then break the cells three times with a homogenizer. Centrifuge the obtained viscous substance at 5,000 rpm, 5¾ for 25 minutes. Collect the supernatant and re-precipitate Soluble in water * under the same conditions Centrifuge. Repeat this step twice. Mix the supernatant and react with trichloroacetic acid (final concentration: 5¾ v / v) and store in 5 scoops overnight. Store the resulting suspension at 5,000 r pm 5 t: 30 minutes in the middle heart. The supernatant was removed, the equal volume of acetone was used to dissolve the precipitate, and the mixture was centrifuged at 5,000 rpm for 15 minutes. The precipitate was dissolved in 500 ml of ethanol / ether, and Dry under reduced pressure. The obtained 52 g of 50% 2H * 13C, 15H-labeled protein in the true air, M 3M code containing 4¾ v / v of hydrothioacetic acid Heat in acid (520 ml) to 100 t :, 4 8 hours. After cooling, slowly pour the hydrolysate into water (520 m 1), and cool in an ice bath, and the resulting mixture Place in an ice bath for about 10 minutes. Centrifuge the obtained cooled solution (RC-3B, 250 mi centrifuge tube, 5 k * 10 minutes). Pump the supernatant M into (200 rol / πι 丨n) Dowex 1X2-400 ion. In the parent resin column (Η + state '2.5 kg) * It is equipped with FM1 laboratory spring model QD on the base. When all the hydrolysate is driven into the column • then People (2 00 -41-paper Zhang applies Chinese National Standard (CNS) _.A4 specification (210X297 mm) 83. 3. 10,000 I --------------'' / L (Please read the precautions on the back before (Fill in this purchase), va printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (in) Diluted sulfuric acid aqueous solution (pH 2.0, 7.5L) and water (11L) into the column. The H * column was pumped to a Dowex 50X8-100 ion exchange resin (0H-state, 2.5 kg) column. Inject 200 ml / rain of diluted ammonia (2¾ v / v, 50L) »from the H + column and the eluent also passed through the 0H_ column. The liquid was collected from the bottom of the 0H-column (2L). TLC analysis ’(Analtech Silica GS plates, n-BuOH: Ac〇H: H20 (2: l: l v / v), developing solution Ning Xidering (1¾ v / v in

Me〇H: Glac AcOH (97: 3 v / v)) showed 50% 2H, 13C, 1SH- labeled Arginine appeared in separations 11-24. After extracting the arginine solution, stop the pump of the pump and connect it directly to the 0H-column. Turn it back on and punch water (14 L) into the 0H-column. Follow up and collect the liquid (2 L). When the 2L separation was continuously collected from the bottom of the column, the diluted aqueous acetic acid solution (0.2525 v / v) was driven into the 0H-column. Continue to run the pump * until no Ningdrin reaction is detected in the eluent. TLC analysis (A na 11 ech S i 1 ica GS p 1 ates, η-B u 0 Η: ~ — AcOH: H2〇 (2: l: lv / v), developing solution Ning Xidlin (1% v / v in

MeOH: Giac AcOH (97: 3 v / v)) shows 50% 2H, 13C, 15N- labeled mixed amino acids (including 50% 2H, 13C, 15H-labeled bran and other amino acids) at 46-58 Creampie regulations. 50% 2H, 13C, 15N-mixed amino acids were separated into light yellow powder by freeze-drying. The 50% 2H, 13C * 1SH-labeled mixed amino acid was dissolved in water (8L), and Dowex 50X8-100 ion exchange resin (acetate state * 500 g) was pumped (50ml / min) from the pump. ) In the column. Collect the liquid (2L).当 -4 2 ~ • l · I—I ——- 1—Q! (Please read the precautions on the back before filling out this page) Order --Θ --- This paper size applies Chinese National Standards (〇 ^) / 4 specifications (2 丨 0 parent 297 mm) 83. 3. 10,000 A7 ^ _________._ B7 ___: _ 5. Description of the invention (+ /) All mixed amino acid solutions are added to the column, and the κ pump beats the water Into the column until all the amino acids have been washed out. TLC analysis (Analtech Silica GS plates, n-BuOH: Ac0H: H20 (2: l: lv / v), developing solution Ningdlin (1¾ v / v in MeOH: Glac AcOH (97: 3 v / v)) shows 50% 2H, 13C, 15H-labeled mixed amino acids (excluding 50% 2H, 13C, 15N-labeled glutamic acid) Appeared in liquid solution 6, which was separated into light yellow powder by freeze drying with a yield of 20.63 g. Μ diluted acetic acid (3%, 6L) to clean the column * 50% 2H * 13C in the separation 7-8, 15N-labeled Trisamino acid is the only amino acid component, which is freeze-dried and separated into white powder. Yield: 2.9 g. Since 13C, 15N and 2H are coupled to 4.19, 3_97, 3.72, 3.48, 3.11, 2.68, 2.26, 2.07 And 1.83 caused DeltaH (D20, 300 MH z) Broad variability. Delta C (D2O, 75 MHz) 177.30 (d), 173.92 (q), 54.20 (d), 53.49 (d), 51.58-50.37 (m), 35.50-34.28 U), 30.36 ( d), 29.64 (d), and 25.92-24.99 (m). ------- Before and after removing 50% 2H '13C, 1SN-labeled glutamic acid, check HPLC for 50% 2H, 13C, respectively, 15N-labeled amino acid mixture (printed by the Consumer Cooperative of the China Standards Bureau of the Ministry of Amine Economy (please read the precautions on the back before filling this page). The acid is derived from o-benzaldehyde derivative, SUpelC0Sil LC-Ιδ, 15x4.6mm column * non-linear gradient from 100% tetrahydrofuran (10%): O2PCU (0.1M): K0Ac (0.2M) to 100% methanol (80%): acetic acid (0.1N * 2 0%) 55 points). Except that the corresponding glutamic acid peak can be completely removed after passing through the acetate resin, its HP LC chromatogram is actually completely different. -43-I Paper size applies Chinese national standard (CNS M4 specification (2 丨 0X297 mm &quot; Ϊ: 83. 3. 10,000 printed by A7, l B7___, Consumer Cooperatives of the Central Bureau of Standards, Ministry of Economic Affairs) 5. Description of the invention (〇 ) Example 10 0 9-Methylmethylchloroformate (3δ8 g, η „〇1) and 1-hydroxysuccinimide (1.73 g, 5mniol) were dissolved in 1,4-dioxolane Rhenium (20 ml) and M triethylamine (2.09 ml, 15 mmol). The resulting suspension was stirred for 15 minutes. 15N-labeled glutamic acid (188 g, Η mmo 丨) and anhydrous sodium carbonate were stirred. (2.54 g, 20 mmol) was dissolved in water, and the resulting solution was added to the 9-pentylmethyl-H-hydroxysuccinyl formic acid. Salt suspension prepared above. The resulting suspension was placed in Stir overnight at room temperature. Allow the solution to evaporate to dry and separate the layer in chloroform (about 200 ml) and water (about 200 ml). Acidify the aqueous layer with concentrated hydrochloric acid to give a value of about 2, then acetic acid Ethyl acetate extraction. The organic layer was dried (anhydrous magnesium sulfate) and dried under reduced pressure to obtain a presumed H-carbomethyl 50% 2H, 13C, 15N-labeled glutamic acid derivative as powder Add paraformaldehyde (2.70 g, 30 mmol) and p-benzylsulfonic acid (a small amount) to the powder obtained as described above, and dissolve it in toluene and allow it to reflux overnight. Allow the solution to cool and place on The residue was reduced to a gum under reduced pressure. This residue -------- layered in ethyl acetate (about 200 ml) and water (about 200 m 1) * The aqueous layer was dried (anhydrous magnesium sulfate ), And evaporated to dryness under reduced pressure to obtain a powdered salamander saliva_ 0 The obtained powder was dissolved in anhydrous tetrahydrofuran (50 m 1) and diisopropylcarbodiimide (2.06 ml, 13.1 mmol), and Before the K dry ice / propane bath was cooled, it was stirred at room temperature for 30 minutes. In addition, 15N-ammonium chloride (2.86 g, 52.5 mmol) was added to a Schlenk test tube, and it was dissolved in dimethylsulfinium ( 25 ml) Medium. Lambda -4 4 ~ This paper size applies Chinese National Standard (CNS). _Λ4 size (210X297 mm) HWo (Please read the precautions on the back before filling in this tile)

-QI I 、 Order—— — .A! I-— ^ n -I----I!. Order printed by the Ministry of Economic Affairs and the Li Bureau Staff Consumer Cooperative A7 B7 V. Description of the invention ((^) Sealing cap 'Immerse this solution in a dry ice / propane bath to freeze. Open the tube cover.' Add sodium hydride (2.1 g, 60% in oil, 52.5 mmol, rinse with hexane and dry with nitrogen) to Tube. Immediately close the test tube, and connect its arm to the bubble generating tube in a condensing conical flask containing a blazolidone derivative. Thaw the dimethylsulfine solution in the Sc h 1 enk test tube, and the resulting 1 5N-ammonia is condensed in the pyrazolidine solution. Wait until all i 5 N _ ammonia is distilled * M clip to close the gas generating tube to close it. Warm the fenazolidone solution at room temperature overnight. Water (2 m 丨) was added to the resulting solution and stirred for 30 minutes. The solution was evaporated to dryness under reduced pressure. The residue was dissolved in ethanol (50 m) and water (100%). M sodium hydroxide (1M, 10.5 ml) reacted with this solvent * and refluxed for 2.5 hours to obtain a hydrazone-protected glutamine derivative. After cooling, hexahydropyridine (1.04 m (1, 10.5 mmol) was reacted with the solution and stirred overnight at room temperature. The resulting solution was evaporated to dryness under reduced pressure, and the layers were separated in chloroform (about 100 ml) and water (about 100 ml). K diacetic acid (Approximately 100 ffll) extract the aqueous layer, and then adjust the pH value to about 7 with glacial acetic acid. Pass the resulting solution through a Dowex 1X2-400 ion exchange resin (NH + state, 20 g) column, and volatilize to dry. M water (approximately 100 ml) was used to dissolve the dried product, and passed it through Do wex 50X8-100 ion exchange resin (acetic acid state * 10 g) * The resulting solution was evaporated to dryness under reduced pressure. Therefore, the obtained 50% 2H , 13C, 15N-Branamine is not crystallized. Part of the obtained amidine is at least 5: 1.5: 1 v / v / v isopropanol: water: glacial acetic acid, which is added to the EM separation Lichroprep DU1 column. Medium (50x2.5 -45 ™ This paper size applies the Chinese National Standard (CNS) &gt; 4 specifications (210x 297 public holidays) 83. 3. 10,000 (read the precautions on the back before filling this page) --- Order The consumer cooperation of the Central Bureau of Standards of the Ministry of Economic Affairs printed A7 B7 V. Description of the invention (C ^ C /) cms) and M 5: 1 V / V / V isopropanol: glacial acetic acid for extraction After mixing the appropriate liquid and liquid phases, the pressure is reduced to 50% 2H, 13C, 15Η-glutamine as the rubber bark. 4.6-This paper size is applicable to Chinese National Standard (CNS) A4 specification (210 × 297) 83) 3. 10,000 (Please read the notes on the back before filling in this page)

Claims (1)

A8 B8 C8 D8 Revised Chinese Patent Application No. 83102456 (September 88) 1. A method for determining the three-dimensional structure information of proteins, which includes the following steps (please read the precautions on the back before filling this page), as in Under the conditions of protein production, mammalian or insect cell cultures that produce the desired protein are grown in a nutrient medium, which contains all the amino acids and absorbable carbohydrate sources necessary for cell growth> Essential minerals and growth factors, wherein all carbon atoms of amino acids in the nutrient medium are substantially UC and all nitrogen atoms of amino acids in the nutrient medium are substantially; Isolate a substantially labeled protein and analyze the protein by KNMR spectroscopy to determine its three-dimensional structure. 2. The method according to item 1 of the scope of patent application, wherein the nutrient medium has a sufficient amount of amino acids with a hydrogen atom of 2Η, which is generated by non-deuterated labeled protein in NMR spectroscopic analysis. Compared with the information, the resolution of the information generated in the NMR spectrum analysis can be enhanced. 3. The method according to item 2 of the scope of patent application, wherein the nutrient medium has a hydrogen atom of amino acid from 20% to 100% as 2H. 4. The method according to item 2 of the scope of patent application, wherein substantially all carbon atoms of the carbohydrate source are 1 3 C, and hydrogen atoms in the carbohydrate source are from 20% to 100% to 2H. Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. A method for determining three-dimensional structure information of a molecule, wherein the first molecule forms a complex with the second molecule, and at least one of the molecules is a protein, which includes steps ⑸ to generate a substance The first molecule labeled with stable isotope with NMR activity is shown on the top; the first molecule is made into a complex with the second molecule, and the complex is analyzed by N MR spectroscopy 'W to determine the first molecule Three-dimensional structure. 6. According to the method in the scope of patent application, item 5 is included before step (b). This paper size applies Chinese National Standard (CNS) A4 specification (210X 297 mm) A8 B8 C8 D8 Revised Chinese Patent Application No. 83102456 (September 88) 1. A method for determining the three-dimensional structure information of proteins, which includes the following steps (please read the precautions on the back before filling this page), as in Under the conditions of protein production, mammalian or insect cell cultures that produce the desired protein are grown in a nutrient medium, which contains all the amino acids and absorbable carbohydrate sources necessary for cell growth> Essential minerals and growth factors, wherein all carbon atoms of amino acids in the nutrient medium are substantially UC and all nitrogen atoms of amino acids in the nutrient medium are substantially; Isolate a substantially labeled protein and analyze the protein by KNMR spectroscopy to determine its three-dimensional structure. 2. The method according to item 1 of the scope of patent application, wherein the nutrient medium has a sufficient amount of amino acids with a hydrogen atom of 2Η, which is generated by non-deuterated labeled protein in NMR spectroscopic analysis. Compared with the information, the resolution of the information generated in the NMR spectrum analysis can be enhanced. 3. The method according to item 2 of the scope of patent application, wherein the nutrient medium has a hydrogen atom of amino acid from 20% to 100% as 2H. 4. The method according to item 2 of the scope of patent application, wherein substantially all carbon atoms of the carbohydrate source are 1 3 C, and hydrogen atoms in the carbohydrate source are from 20% to 100% to 2H. Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. A method for determining three-dimensional structure information of a molecule, wherein the first molecule forms a complex with the second molecule, and at least one of the molecules is a protein, which includes steps ⑸ to generate a substance The first molecule labeled with stable isotope with NMR activity is shown on the top; the first molecule is made into a complex with the second molecule, and the complex is analyzed by N MR spectroscopy 'W to determine the first molecule Three-dimensional structure. 6. According to the method in the scope of the patent application, before step (b), this paper includes the standard of Chinese paper (CNS) A4 (210X 297 mm). The paper is printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs. A8 B8 C8, D8 _ 6. The scope of patent application, essentially marking the second molecule as deuterium. 7. The method according to item 5 of the scope of the patent application, wherein the first molecule is a protein, which can be produced by the following methods, including, (a &gt; Under the conditions of protein production, mammalian or insect cells that are to be investigated for proteins will be produced The culture is grown in a nutrient medium, which contains all the amino acids and absorbable carbohydrate sources necessary for cell growth, 'essential minerals and growth factors, of which amino acids are essentially K isotope with NMR-active isotopes And (b &gt; Isolate a substantially labeled protein from a nutrient medium. 8. The method according to item 7 of the scope of the patent application, wherein the second molecule is a protein and can be produced by the following method, its steps Including (a) under the conditions of protein production, mammalian or insect cells that can produce the protein to be investigated are grown in a nutrient medium, which contains all the amino acids and absorbable carbohydrate sources necessary for cell growth , Must be minerals and growth factors, in which the amino acids are substantially marked; and the belly is separated from the nutrient medium The labeled protein is qualitative. 9. According to the method in the scope of patent application No. 8 wherein the first molecule is a protein, it can be produced by the following method. The steps include the following conditions: To explore the growth of mammalian or insect cells of proteins in a nutrient medium, this medium contains all the amino acids and absorbable carbohydrate sources necessary for cell growth, essential minerals and growth factors, of which, in essence, amines The basic acid is labeled with an NMR-active isotope; and &lt; bi separates the substantially labeled protein from the nutrient medium. 10. According to the method of item 5, 6, 7, 8 or 9 of the scope of patent application, Among them, the NMR paper size is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) (Please read the precautions on the back before filling this page) I ^ -------- Order ----- Ministry of Economic Affairs Printed by the Consumer Standards Cooperative of the Central Bureau of Standards A8 B8 C8 D8 6. The isotope of the activity in the scope of patent application is 1 3 C. 11. The method according to item 5, 6, 7, 8 or 9 of the scope of patent application, of which The first molecule is double labeled with 1 3 C and 1 5 N. 12. According to the method of item 9 in the scope of the patent application, the isotope with NMR activity is 1 3C or 1 3C and 1 5N, and is used to prepare the first molecule. In a molecular nutrient medium, a sufficient amount of hydrogen atoms of the amino acid is 2H '. Compared with the NMR spectrum signal of the labeled protein, which is enhanced by K, it is analyzed by the NMK spectrum analysis signal. 13. The method according to item 12 of the scope of patent application, wherein the amino acid in the nutrient medium used to prepare the first molecule has a hydrogen atom of 2H from 20% to 100%. 14. A nutrient medium capable of supplying mammalian or migratory cells for growth, which contains all amino acids, absorbable carbohydrates, and essential minerals and growth factors required for cell growth. The amino acids and any other substrates used in protein synthesis are labeled with M 13C and 1 5 N. 15. The nutrient medium according to item 14 of the scope of the patent application, wherein the source of carbohydrate K 1 3 C is substantially indicated. 16. The nutrient medium according to item 14 or 15 of the scope of patent application *, wherein the hydrogen atom of the amino acid in the nutrient medium is from 20% to 100% is 2H 〇17. The nutrition according to item 16 of the scope of patent application In the culture medium, cysteine substantially labeled with 1 3 C and 15 N is added, and the hydrogen atom from 20% to 100% is 2H. 1 8 · Nutritional medium according to item 16 of the scope of the patent application, in which the actual paper size is added to the Chinese National Standard (CNS) M specification (210 × 297 mm) (Please read the precautions on the back before filling this page) Order Α8 Β8 C8 D8 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs. 6. The scope of patent application for glutamine is marked by K 13C and 13N, and its hydrogen atom from 20% to 100% is 2 Η. 19. The nutrient medium according to item 16 of the scope of the patent application, wherein aspartame is actually added with K 1SC and 15N as the label, and the hydrogen atom from 20% to 100% is 2%. 20. The nutrient medium according to item 14 of the scope of the patent application, wherein the amino acid is substantially labeled K 2 Η. .21. The nutrient medium according to item 25 of the scope of the patent application, wherein the source of the carbohydrate is substantially K 2Η. 22. A method for producing a substantially isotopically labeled amino acid mixture, including ⑶ growing microorganisms in a nutrient medium, wherein all carbons of any substrates used by microorganisms for protein biosynthesis are substantially 13C, and all nitrogens of any substrate used by microorganisms for protein biosynthesis are 15N in nature, or all hydrogen of any substrates used by microorganisms for protein biosynthesis is substantially 2 Η; ( b) collecting protein-containing liquid fractions from the culture broth; ® hydrolyzing the protein M in the presence of a thiohydrogen source agent under acidic, non-oxidizing conditions to produce a crude product of the amino acid mixture; (d) the amino acid mixture The crude product K was purified by cation exchange resin chromatography, and K was used to produce a partially purified amino acid mixture; (e) the partially purified amino acid mixture K was purified by anion exchange resin chromatography. M produces a purified amino acid mixture; and (f) M isotope-labeled cysteine is added to the purified amino acid stick compound, and the isotope or the isotope mixture and the original Medium were identical, and wherein the amount of cysteine added protein may be a sufficient supply Κ mammalian or insect cells is generated. (Please read the precautions on the back before filling out this page) b Binding. Binding · This paper size adopts Chinese National Standard (CNS) A4 specification (210X297 mm) ABCD b8iiv3 ττ, patent application and application 23. According to patent application The method according to item 22, wherein the nutrient medium and cysteine K 13C are labeled. 24. The method according to item 22 of the scope of patent application, wherein the nutrient medium and cysteine M 1 5 N are marked. 25. The method according to item 22 of the scope of patent application, wherein the nutrient medium and cysteine K are marked. 26. The method according to item 22 of the scope of patent application, wherein the nutrient medium and cysteine M13C are double labeled. 27. The method according to item 26 of the scope of patent application, wherein the nutrient medium and cysteine are marked with M 2Η, wherein the purified amino acid mixture and the hydrogen atom of cysteine are composed of 20% To 100% is 2%. 28. The method according to item 27 of the scope of patent application, which further comprises adding substantially glutamine labeled with M 1 3 C and 1 5 N to the purified amino acid mixture, and the hydrogen atom therein is from 20% to 100% is 2%. 29. The method according to item 28 of the scope of patent application, which further comprises adding asparagine substantially labeled K 1 3 C and 1 5 N to the purified amino acid mixture, and wherein the hydrogen atom is 20% To 100% is 2Η. 30. The method according to item 22 of the scope of patent application, wherein the microorganism is a microfamily, and the carbon source contained in the nutrient medium is carbon dioxide labeled 1 3 C-. 31. The method according to item 22 of the scope of the patent application, wherein the emblem organism is microalgae (m i c r 0 a 1 g a 1), and the nutritional medium contains a 15 N-labeled inorganic nitrogen source. 32. The method according to item 22 of the scope of patent application, wherein the microorganism is microalgae, and the carbon source contained in the nutrient medium is 13C-labeled. This paper scale is applicable to the Chinese National Standard (CNS) A4 specification (210X297). (Mm) (Please read the precautions on the back before filling out this page): --J --------------, 玎 — —:: --- Staff Consumption of Central Bureau of Standards, Ministry of Economic Affairs The cooperative printed S8ii78 A8 B8 C8 D8. The emblem 20 in the 2H is. Its homogeneous source, the basis of the water and nitrogen method is not the basis of the term of the cultivation of the 22nd standard N-culture 15 Fanying economic benefits and special carbon, please apply for application and oxygen data, two roots隹 0 Yang's use of the law's term 2 2 perimeter. Paradigm H &quot; Designed specifically for the purpose of youth-mouth 3 »Shen Shu according to the law in the use of the law to change the root of the cross to the yin 2 2 2. Circumstance Fan _ ϋ 禾 禾 ο Designed for. Please ask Zhishen Shu to change its roots to the law of the project Liyang base, which is made from amines, substances (d) The dyeing solution can be quickly stained and dissolved. Sexual acid and acid's removal of water and extraction of the 34th mass of the white perimeter egg column fan will control the tube, the lipid-specific liquid tree, please dissolve the exchange of acid roots. The 36th column tube in the red item mentioned by the French formulator. To change Fan Jiaolizi's special departure, please ask Yang Shen to replace it according to the tube acid (please read the precautions on the back before filling this page) for the column (e) Step Yu Qili crosses the egg and dissolves the solubility of 0 yin so that the alkali can be used in the middle of the column to change the cross tube to cross the yin and yin. The acid solution is dyed by amine to dissolve the sexual solution and sex. Acids and bases are beneficial and decontaminated. ”Water removal of quality and whitening include the standard amino acid and cysteine of the standard display unit. The same preparation and production of seeds are produced by the central government bureau of the Ministry of Economic Affairs and printed by the consumer cooperative. Most of the peptides in the amino acid cysteine have the following advantages, which should be the same as in the formula SI4 of the standard -I element 4 &amp; From this paper size, the Chinese National Standard (CNS) A4 specification (210 X 297 mm) is applicable ^ 81178 Α8 Β8 C8 D8 6. Scope of patent application CH2COOH where R and R 'are each hydrogen; (bJ (i) hydrolysis of the polypeptide to produce an amino acid mixture containing a protected thioether; and (ii) using ion exchange column chromatography, Protected thioethers are separated from the amino acid mixture Ϊ5; and ⑵ deprotects the protected sulfide to produce isotope-labeled cysteine. 39.-a preparation of isotope-labeled cysteine, where Substantially all carbon atoms are 13C, and substantially all nitrogen atoms are 15N, and from 20% to 100% hydrogen atoms are 2H. 40. A method for preparing isotope-labeled glutamine or aspartamine 'It includes: (3) reacting isotope-labeled glutamic acid or aspartic acid with N-fluorenylmethyl to generate N-protected amino acids of the following formula (please read the precautions on the back before filling in This page) Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs I X-RN-CHI i CH,) c R〇y% This paper is used for New Zealand + National Standards (CNS) Λ4 Listening (210X297K: t) A8 B8 C8 D8 d8li78 Patent Application Park &lt; b) Make N-protected amino acid and paraformaldehyde in a catalytic amount In the presence of a strong acid, the reaction M produces oxazolidinone of the following formula, (° Υ ° XN-CH I (CHJn I human, Ή Ο Ο (〇) under hydrazine-forming conditions, the azrididone is reacted with ammonia, μ Cycloperamide derivatives of the following formula (please read the notes on the back before filling this page) 〇 、 〇 XN-CH I ^ Η,) η I .Η, N Ν0 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (d) Hydrolyze the cyclic amide derivative to form Ν-protected amino acid H0W 〇c X-HN-CH I (strict) π A Η〆V &lt; e) deprotect N- with a protected amino acid, and generate an isotope-labeled paper. The paper size applies Chinese National Standard (CNS) A4 specifications (210 X 297 mm) A8 B8 C8 D8 ^ 81178 6. The scope of patent application: Asparagine. Where ri is 1 or 2 and X is an amine protecting group. 41. A preparation of glutamine, wherein substantially all carbon atoms are 13C, and substantially all nitrogen atoms are, and the hydrogen atom from 20% to 100% is 2%. 42. A preparation of asparagine, wherein substantially all carbon atoms are 13C, and substantially all nitrogen atoms are. 43. The preparation according to item 42 of the scope of patent application, wherein the hydrogen atom from 20% to 100% is 2H. (Please read the notes on the back before filling this page) Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs This paper is sized for the Chinese National Standard (CNS) A4 (210X297 mm)