WO2016169307A1 - 肌酸激酶突变体、基因及突变体的应用 - Google Patents

肌酸激酶突变体、基因及突变体的应用 Download PDF

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WO2016169307A1
WO2016169307A1 PCT/CN2016/070724 CN2016070724W WO2016169307A1 WO 2016169307 A1 WO2016169307 A1 WO 2016169307A1 CN 2016070724 W CN2016070724 W CN 2016070724W WO 2016169307 A1 WO2016169307 A1 WO 2016169307A1
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creatine kinase
creatine
mutation
sequence
ckm
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French (fr)
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傅荣昭
付荣昕
赵丽青
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邦泰生物工程(深圳)有限公司
江西安泽麦生物科技有限公司
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Priority to CN201680003940.3A priority Critical patent/CN107109379B/zh
Publication of WO2016169307A1 publication Critical patent/WO2016169307A1/zh

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    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
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    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1223—Phosphotransferases with a nitrogenous group as acceptor (2.7.3)
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    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/03—Phosphotransferases with a nitrogenous group as acceptor (2.7.3)
    • C12Y207/03002—Creatine kinase (2.7.3.2)

Definitions

  • the present invention relates to the field of molecular biology and biotechnology, and in particular to the use of creatine kinase mutants, genes and mutants.
  • Phosphocretinine a high-energy phosphate compound in muscle or other excitable tissues such as the brain and nerves, is a temporary storage form of high-energy phosphate groups.
  • the role of creatine phosphate is very much, and its main effects are as follows: (1) Myocardial protective agent: Creatine phosphate is widely distributed in various tissues of the body, 90% in muscle tissue, and creatine phosphate is used to maintain ATP levels. The creatine phosphate protects the muscle fiber membrane from ischemic damage and maintains the cellular nucleic acid reserve by opening the synthetic pathway and reducing decomposition. Cardiac protection for cardioplegia and other conditions of myocardial metabolic distress.
  • ATP adenosine triphosphate
  • a creatine kinase mutant wherein the sequence 2 of the sequence listing is a reference sequence having at least one mutation selected from the 100th, 121st, and 33rd positions, and the adenosine triphosphate and creatine are The substrate has at least 50% more creatine kinase catalytic activity than the parent.
  • a gene comprising a nucleotide sequence encoding the above-described creatine kinase mutant.
  • the present invention obtains a series of highly catalytically active creatine kinase mutants by site-directed mutagenesis of the Oryctolagus cuniculus creatine kinase gene, PCR amplification, insertion of an appropriate vector, and subsequent screening on LB medium.
  • the mutant can use creatine and adenosine triphosphate (ATP) as a substrate to efficiently catalyze the production of creatine phosphate.
  • ATP adenosine triphosphate
  • FIG. 1 is a polyacrylamide gel electrophoresis pattern of a creatine kinase parent and a mutant F100Y.
  • the three lanes from left to right are protein molecular weight standards, parent creatine kinase crude protein (A), muscle.
  • the acid kinase mutant F100Y crude protein (B), the arrow indicates the position of the enzyme of interest.
  • the present invention provides the use of creatine kinase mutants, genes and mutants, and the present invention will be further described in detail below in order to clarify and clarify the objects, technical solutions and effects of the present invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
  • a creatine kinase mutant of the present invention which has the following sequence 2 as a reference sequence, has at least one mutation selected from the 100th, 121st, and 33rd positions, and is adenosine triphosphate (ATP) And creatine as a substrate which has at least 50% higher creatine kinase catalytic activity than the parent.
  • the 100th phenylalanine (Phe) in the parent sequence is mutated to tyrosine (Tyr); the 121th leucine (Leu) mutation in the parent sequence Aspartic acid (Asp); and/or the proline (Val) at position 33 in the parent sequence is mutated to alanine (Ala).
  • mutants can be purified by conventional Histag and have high catalytic activity.
  • a mutant F100Y having a single point mutation has a higher specific activity than the parent.
  • the invention provides a gene comprising a nucleotide sequence encoding a creatine kinase mutant of the invention.
  • the invention also relates to the use of a creatine kinase mutant for the preparation of creatine phosphate using adenosine triphosphate (ATP) and creatine as substrates.
  • ATP adenosine triphosphate
  • the creatine kinase mutant may be used in the form of a crude enzyme without purification, or may be a partially purified or fully purified enzyme. If desired, the creatine mutant of the present invention can also be made into a solid phase enzyme or solid phase cell solidified enzyme using a curing technique known in the art.
  • a vector plasmid containing the parent creatine kinase gene is first constructed, and then the site of the site-directed mutagenesis and the amino acid species after the mutation are set, and then the appropriate primer is synthesized, and the parent is contained.
  • the vector plasmid of the creatine kinase gene is used as a template, and the DNA fragment is PCR-amplified, the amplified DNA fragment is assembled, and the full-length mutant gene is PCR-amplified.
  • a full-length mutant gene can also be synthesized, and then the full-length mutant gene can be cloned into an appropriate vector and transformed into an appropriate host cell, and a positive clone having creatine kinase activity can be selected by culture. Finally, plasmid DNA was extracted from the positive clones and subjected to DNA sequence analysis to determine the introduced mutation.
  • any appropriate carrier can be employed.
  • suitable vectors include, but are not limited to, prokaryotic expression vectors, such as pRSET and pES21, and the like; include, but are not limited to, cloning vectors such as PUC18/19 and pBluscript-SK.
  • the obtained creatine kinase mutant gene can be expressed in a prokaryotic or eukaryotic cell, or can be achieved by any other suitable method known in the art. Expressed extracellularly in prokaryotic or eukaryotic cells.
  • the host cell of the vector is a prokaryotic cell or a eukaryotic cell.
  • the prokaryotic cells include, but are not limited to, E. coli.
  • the eukaryotic cells include, but are not limited to, Saccharomyces cerevisiae and Pichia pastoris.
  • parent refers to from Oryctolagus cuniculus.
  • the creatine kinase has the nucleotide sequence shown in SEQ ID NO: 1 (see GenBank NM_001082239) and the amino acid sequence as shown in SEQ ID NO: 2 (see GenBank NP_001075708).
  • reference sequence when it is a nucleotide sequence, refers to sequence 1 in the sequence listing, and when it is an amino acid sequence, it refers to sequence 2 in the sequence listing.
  • reference sequence and the mutated creatine kinase sequence are sorted and compared, they can be performed manually or by computer (currently More computer software available, such as the CLUSTALW program, etc.).
  • creatine kinase mutant refers to a sequence in which the amino acid sequence shown in SEQ ID NO: 2 in the Sequence Listing is used as a reference sequence, and is selected from the 100th, 121st, and 33rd positions. At least one mutation, and the production of creatine phosphate using creatine and adenosine triphosphate (ATP) as a substrate, which has at least 50% higher creatine kinase catalytic activity than the parent.
  • ATP adenosine triphosphate
  • the variant of the creatine kinase mutant includes a conservative substitution form of the other amino acid sequences other than the 100th, 121st, and 33rd positions in the amino acid sequence shown in SEQ ID NO: 2,
  • the absence of one or several amino acid forms, the amino-terminal truncated form, the carboxy-terminal truncated form, and some or all of the tandem repeats of SEQ ID NO: 2 are also included within the scope of the invention.
  • the three-letter or one-letter expression of the amino acid used in the invention employs the amino acid code specified by IUPAC (Eur. J. Biochem., 138: 9-37, 1984).
  • mutants prepared by the present invention and their properties are described below using specific examples.
  • the following examples are intended to illustrate the invention and are not to be considered as limiting the scope of the invention. If no specific conditions are specified in the examples, they are carried out under the usual conditions or conditions recommended by the manufacturer.
  • Example 1 Amplification and cloning of a creatine kinase-encoding gene
  • Amplification conditions were: 20 mM Tris-HCl (pH 8. 8), 10 mM KC1, 10 mM (NH4) 2S04, 2 mM MgS04, 0.1% Triton X-100, 50 mM dATP, 50 mM dTTP, 50 mM dCTP, 50 mM dGTP, 400 nM bow
  • the PCR amplification reaction procedure was: 95 ° C for 3 minutes, 35 cycles: 95 ° C for 50 seconds, 50 ° C for 30 seconds, and 72 ° C for 1 minute, and finally 72 ° C for 10 minutes.
  • the amplified product was digested with restriction endonucleases Ndel and BamHI and ligated with the vector pRSET-A (derived from Invitrogen, USA) digested with the same restriction endonucleases Ndel and BamHI to obtain plasmid pRSET-ckm.
  • the nucleotide sequence of the cloned creatine kinase was determined by DNA sequencing, and is shown in Sequence 1 in the sequence listing. The corresponding amino acid sequence is Sequence 2 in the Sequence Listing.
  • primers 100YF and 100YR were designed using plasmid pRSET-ckm (see Example 1) as a template ( See Table 1).
  • the F-YR fragment was amplified with primer pairs ckm-F and 100YR, and the YF-R fragment was amplified with primer pairs 100YF and ckm_R.
  • the amplification reaction conditions were: 20 mM Tris-HCl (pH 8. 8), 10 mM KC1, 10 mM
  • nM primer 100YF and 400 nM primer ckm-R the reaction volume was adjusted to 50 ⁇ l with sterile water.
  • the PCR amplification reaction procedure was: 95 ° C for 3 minutes, 35 cycles: 95 ° C for 50 seconds, 52 ° C for 30 seconds, and 72 ° C for 3 minutes, and finally 72 ° C for 5 minutes.
  • the mixture was separated by electrophoresis on a 1% agarose gel and recovered by a commercial kit to obtain an F-YR fragment and a YF-R fragment, respectively.
  • the full length gene is then amplified.
  • the amplification reaction conditions were: 20 mM Tris-HCl (pH 8.
  • the PCR amplification reaction procedure was: 95 ° C for 3 minutes, 35 cycles: 95 ° C for 50 seconds, 52 ° C for 30 seconds, and 72 ° C for 3 minutes, and finally 72 ° C for 5 minutes. It was separated by 1% agarose gel electrophoresis and recovered by a commercial kit to obtain a full-length mutant gene F100Y.
  • the F100Y fragment was recovered, digested and recovered, and ligated with the vector pRSET-A (refer to Example 1) to obtain plasmid pRSET-F100Y. Transfer of plasmid pRSET-FlOO Y to competent bacterial cells E. col i BL21 o
  • the point mutations introduced were confirmed by DNA sequencing.
  • the amino acid sequence of F100Y is shown in SEQ ID NO: 3 in the Sequence Listing.
  • Example 3 Site-directed mutagenesis of creatine kinase mutant site 121
  • primer pairs 121DF and 121DR were designed using plasmid pRSET-ckm (see Example 1) as a template ( See Table 1).
  • the DF-R fragment was amplified by amplifying the F-DR fragment with the primer pair ckm-F and 121DR, and the primer pair 121DF and ckm_R.
  • the specific sequences of the primers ckm-F and ckm-R are shown in Table 1.
  • the amplification reaction conditions were: 20 mM Tris-HCl (pH 8. 8), 10 mM KC1, 10 mM (NH4) 2S04, 2 mM MgS04, 0.1% Triton
  • ckm-R 1. 5 U Pfu DNA polymerase (Promega, USA), 20 ng pRSET-ckm, adjust the reaction volume to 50 ⁇ l with sterile water.
  • the PCR amplification reaction procedure was: 95 ° C for 3 minutes, 35 cycles: 95 ° C for 50 seconds, 52 ° C for 30 seconds, and 72 ° C for 3 minutes, and finally 72 ° C for 5 minutes.
  • the mixture was separated by electrophoresis on a 1% agarose gel and recovered by a commercial kit to obtain an F-DR fragment and a DF-R fragment, respectively.
  • the full length gene is then amplified.
  • the amplification reaction conditions were: 20 mM Tris-HCl (pH 8.
  • the agarose gel was separated by electrophoresis and recovered by a commercial kit to obtain a full-length mutant gene L121D.
  • L121D was ligated to the vector pRSET-A (refer to Example 1) to obtain plasmid pRSET-L121D.
  • the plasmid pRSET_L121D was transformed into a competent bacterial cell E. coli BL21 o. The point mutation introduced was confirmed by DNA sequencing.
  • the amino acid sequence of L121D is shown in Sequence 4 in the Sequence Listing.
  • primers 33AF and 33AR were designed using plasmid pRSET-ckm (see Example 1) as a template ( See Table 1).
  • the F-AR fragment was amplified with primer pairs ckm-F and 33AR, and the primer pair 33AF and Pckm-R was used to amplify the AF-R fragment.
  • the specific sequences of the primers ckm-F and ckm-R are shown in Table 1.
  • the amplification reaction conditions were: 20 mM Tris-HCl (pH 8. 8), 10 mM KC1, 10 mM (NH4) 2S04, 2 mM MgS04, 0.1% Triton X-100, 50 mM dATP, 50 mM dTTP, 50 mM dCTP, 50 mM dGTP, 400 nM bow
  • the full length gene is then amplified.
  • the amplification reaction conditions are: 20 mM
  • Tris-HCl pH 8. 8
  • 10 mM KC1 10 mM
  • 10 mM (NH4) 2S04 2 mM MgS04, 0.1%
  • Triton X-100 50 mM dATP, 50 mM dTTP, 50 mM dCTP, 50 mM dGTP, 400 nM primer ckm-F and 400 nM ckm-R, 1. 5 U Pfu DNA polymerase, 20 ng F-AR fragment and 20 ng of the AF-R fragment, the reaction volume was adjusted to 50 ⁇ l with sterile water.
  • the PCR amplification reaction procedure was: 95 ° C for 3 minutes, 35 cycles of 95 ° C for 50 seconds, 52 ° C for 30 seconds, and 72 ° C for 3 minutes, and finally 72 ° C for 5 minutes.
  • V33A was ligated to the vector pRSET-A to obtain plasmid pRSET-V33A.
  • the plasmid pRSET_V33A was transferred to competent bacterial cells E. col i BL21 o
  • the point mutation introduced was confirmed by DNA sequencing.
  • the amino acid sequence of the resulting mutant is shown in SEQ ID NO: 5 in the list.
  • BL21 was cultured on a Luria broth (LB) plate (containing 50 mg/L kanamycin) for 24 hours at 37 °C. Inoculation A single clone was cultured in 5 ml of LB liquid medium (containing 50 mg/L kanamycin) at 30 ° C for 20-24 hours. The cells were collected by centrifugation and suspended in 1 ml of 100 mM Tris-HCl buffer (pH 7.5). The bacterial cells are then lysed by ultrasonic waves. Centrifuge (10 ° C, 17, 800 g, 10 minutes) and collect the supernatant, which is the crude protein (or crude extract).
  • LB Luria broth
  • Figure 1 A shows the results of polyacrylamide gel electrophoresis of the recombinant parent creatine kinase crude protein, indicating that creatine kinase (target band size is about 42 kD) is expressed in E. coli BL21.
  • the plasmid pRSET-FlOOY containing the creatine kinase gene was transformed into competent bacterial cells E. col i BL21, cultured on a Luria broth (LB) plate (containing 50 mg/L kanamycin) for 24 hours at 37 °C.
  • a single clone was inoculated in 5 ml of LB liquid medium (containing 50 mg/L kanamycin) and incubated at 30 ° C for 20-24 hours.
  • the cells were collected by centrifugation and suspended in 1 ml of 100 mM Tris-HCl buffer (pH 7.5).
  • the bacterial cells are then lysed with ultrasound. Centrifuge (10 ° C, 17, 800 g, 10 minutes) and collect the supernatant, which is the crude protein (or crude extract).
  • Figure 1B shows the results of polyacrylamide gel electrophoresis of recombinant creatine kinase mutant F100Y crude protein, indicating that creatine kinase (target band size is about 42kD) is higher in E. coli BL21. The level of expression.
  • Example 7 Determination of the activity of the parent creatine kinase
  • the pH was adjusted to 7.5. 400 ⁇ l of the substrate solution was taken, and then 100 ⁇ l of the parent creatine kinase was added, and the reaction was carried out at 37 ° C for 10 minutes. Centrifuge (10 ° C, 17, 800 g, 15 minutes) and collect the supernatant. The content of creatine phosphate in the obtained supernatant was measured by high pressure liquid chromatography (HPLC). The enzyme protein concentration was measured by SDS polyacrylamide gel electrophoresis, and the results are shown in Table 2. One unit enzyme specific activity is defined as the amount of enzyme required to convert one micromole of creatine to creatine phosphate per minute under the above conditions. The specific activity of creatine kinase is 3. 5U/mg 0
  • Example 8 Determination of creatine kinase mutant activity
  • the creatine kinase crude enzyme was diluted with a washing enzyme buffer (0.02 M Tris-HCl/O. OOlM EDTA, pH 7.0 solution) to a protein content of 5-10 mg/ml.
  • the enzyme dilution was mixed with PB solution (2.0 mol/L potassium dihydrogen phosphate, pH 7.5) in equal volume, and the immobilized enzyme carrier LX-3000 (10 mg enzyme/g carrier) was added to the shaker (rotation speed 100 rpm).
  • the reaction was carried out at 25 ° C for 20 hours. After the reaction was completed, it was filtered with a filter bag and washed with a washing enzyme buffer for 5-6 times to obtain immobilized creatine kinase.

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Abstract

一种肌酸激酶突变体、基因及突变体的应用，该突变体以所附的序列2为参考序列，其具有选自第100位、第121位和第33位的至少一个突变，并且以肌酸和三磷酸腺苷二钠(ATP)为底物其具有比亲本高出至少50％的肌酸激酶催化活性。该肌酸激酶突变体可用于生产磷酸肌酸。

Description

肌酸激酶突变体、 基因及突变体的应用 技术领域
[0001] 本发明涉及分子生物学与生物技术领域， 具体地说， 涉及肌酸激酶突变体、 基 因及突变体的应用。
背景技术
[0002] 磷酸肌酸 （phosphocreatine ) ， 是在肌肉或其他可兴奋组织 （如脑和神经） 中的一种高能磷酸化合物， 是高能磷酸基的暂时贮存形式。 磷酸肌酸的作用非 常多， 其主要作用有以下七点： （1 ) 心肌保护剂： 磷酸肌酸广泛地分布于身体 各组织， 90%在肌肉组织中， 磷酸肌酸是用来维持 ATP水平的， 磷酸肌酸通过开 放合成通路和减少分解作用， 保护肌纤维膜免受缺血损害并维持细胞的核酸储 备。 临床用于心麻痹症的心脏保护及心肌代谢窘迫的其他状况。 适用于心肌缺 血、 肥厚、 心梗及心衰的治疗 （辅助治疗） ， 亦可用作各种心脏手术； （2 ) 缓 冲肌肉中酸性物质突然增高； （3 ) 磷酸肌酸 (CP)还参与能量运输， 即把能量从 线粒体运载到肌肉的其它部位； （4) 运动员首选的运补剂： 磷酸肌酸对于增加 运动员的体能， 提高运动成绩有明显的效果、 安全有效， 无副作用。 在比赛中 ， 磷酸肌酸 (CP)水平的增加能提高训练和比赛成绩； （5 ) ATP的贮存形式： 磷 酸肌酸可以把高能磷酸转移给 ADP生成 ATP，因此磷酸肌酸是 ATP的贮存形式； （6 ) 与40？作用而产生 ATP: 当体内肌酸磷酸激酶降低至零之前，脑组织缺氧时， 磷 酸肌酸均能与 ADP作用而产生 ATP; ( 7 ) 缓冲剂的作用： 磷酸肌酸除提供能量外 ， 在大强度练习中还可以对控制肌肉中的酸性物质起缓冲剂的作用。 这是因为 磷酸肌酸在合成 ATP过程中需要消耗大强度练习中堆积在肌肉中乳酸释放出来的 氢离子， 而肌肉中氢离子过多会阻碍肌肉收缩， 所以磷酸肌酸能起缓冲作用并 推迟疲劳的出现。
[0003] 当前市场上的原料药磷酸肌酸是化学合成的， 由于合成过程中需要用到有毒且 易燃原料， 政府不再颁发生产环保批文， 严重引影响磷酸肌酸的应用。
[0004] 随着生物科技的飞速发展， 人类比以往任何时候更加关注自身的健康长寿和环 境问题， 2015年初， 新的 《环境保护法》 正式施行， 这部被誉为史上最严厉的 环境保护法的出台， 也反应了政府大力改进中国环保现状的决心。 2015年 3月中 央政治局会议首提 "绿色化" ： "四化"变 "五化" 。 "绿色化"是指 "科技 含量高、 资源消耗低、 环境污染少的产业结构和生产方式" 。 颠覆传统 "高污 染、 高耗能、 高碳排放"化学合成产业的绿色生物合成将成为下一轮产业经济 新的增长点， 并将成为生物经济的发动机。
[0005] 尽管通过生物催化技术生产磷酸肌酸不仅高效且具有低碳环保， 具有很强的竞 争力， 但用于生物催化技术生产磷酸肌酸的肌酸激酶活力较低， 影响着该技术 的工业化应用。
[0006] 因此， 提高肌酸激酶催化活力是降低绿色生物合成磷酸肌酸生产成本的关键因 素。
[0007] 因此， 现有技术还有待于改进和发展。
技术问题
[0008] 本发明的目的在于提供高催化活性的肌酸激酶突变体。 本发明的另一目的还在 于提供含有编码本发明所述的肌酸激酶突变体的基因。 本发明的再一目的在于 将将：本发明的突变体应用于以肌酸和三磷酸腺苷 (ATP)为底物， 生产磷酸肌酸 t 问题的解决方案
技术解决方案
[0009] 本发明的技术方案如下：
[0010] 一种肌酸激酶突变体， 其中， 以序列表的序列 2为参考序列， 其具有选自第 100 位、 第 121位和第 33位的至少一个突变， 并且以三磷酸腺苷和肌酸为底物时其具 有比亲本高出至少 50%的肌酸激酶催化活性。
[0011] 所述的肌酸激酶突变体， 其中， 其中第 100位的苯丙氨酸突变为酪氨酸。
[0012] 所述的肌酸激酶突变体， 其中， 其中第 121位的亮氨酸突变为天冬氨酸。
[0013] 所述的肌酸激酶突变体， 其中， 其中第 33位的缬氨酸突变为丙氨酸。
[0014] 所述的肌酸激酶突变体， 其中， 其具有所附序列表中 SEQ ID NO. : 3至 SEQ ID
NO. : 5之一所示的氨基酸序列。
[0015] 一种基因， 其中， 其含有编码上述的肌酸激酶突变体的核苷酸序列。 [0016] 一种如上述的肌酸激酶突变体的应用， 其中， 将其应用于以三磷酸腺苷和肌酸 为底物制备磷酸肌酸的过程中。
发明的有益效果
有益效果
[0017] 本发明通过对 Oryctolagus cuniculus 肌酸激酶基因进行定点突变， PCR扩增 后插入适当的载体， 随后在 LB培养基上筛选， 从而获得了一系列具高催化活性 的肌酸激酶突变体， 该突变体能以肌酸和三磷酸腺苷 (ATP)为底物， 可高效率催 化生成磷酸肌酸。
对附图的简要说明
附图说明
[0018] 图 1为 肌酸激酶亲本与突变体 F100Y之聚丙烯酰胺凝胶电泳图， 图中从左至右 的三条泳道依次为蛋白分子量标准、 亲本肌酸激酶粗提蛋白（A)、 肌酸激酶突变 体 F100Y粗提蛋白（B) ， 箭头指示目的酶位置。
发明实施例
本发明的实施方式
[0019] 本发明提供肌酸激酶突变体、 基因及突变体的应用， 为使本发明的目的、 技术 方案及效果更加清楚、 明确， 以下对本发明进一步详细说明。 应当理解， 此处 所描述的具体实施例仅仅用以解释本发明， 并不用于限定本发明。
[0020] 本发明一种肌酸激酶突变体， 其以所附的序列 2为参考序列， 其具有选自第 100 位、 第 121位和第 33位的至少一个突变， 并且以三磷酸腺苷 (ATP)和肌酸为底物 其具有比亲本高出至少 50%的肌酸激酶催化活性。
[0021] 优选的是， 所述亲本序列中的第 100位的苯丙氨酸 (Phe)突变为酪氨酸 (Tyr)； 所述亲本序列中的第 121位的亮氨酸 (Leu)突变成天冬氨酸 (Asp) ; 和 /或所述亲 本序列中的第 33位的缬氨酸 (Val)突变成丙氨酸 (Ala)。
[0022] 这些突变体可以通过常规 Histag纯化， 并具有高的催化活性。 例如， 在本发明 获得的一系列突变体中， 一个具有单点突变的突变体 F100Y的比活性较亲本高出
111%。 [0023] 另一方面， 本发明还提供了一种基因， 其含有编码本发明的肌酸激酶突变体的 核苷酸序列。
[0024] 又一方面， 本发明还涉及肌酸激酶突变体的应用， 其应用于以三磷酸腺苷 (ATP )和肌酸为底物制备磷酸肌酸。
[0025] 所述的肌酸激酶突变体可以未经纯化以粗酶形式使用， 也可以是经部分纯化的 或完全纯化的酶。 如果需要， 还可利用本领域已知的固化技术将本发明肌酸激 酶突变体制成固相酶或固相细胞形式的固化酶。
[0026] 为了获得本发明的突变体， 先构建含有亲本肌酸激酶基因的载体质粒， 然后设 定定点突变的位点以及突变后的氨基酸种类， 再合成适当的引物， 以所述的含 亲本肌酸激酶基因的载体质粒为模板， PCR扩增 DNA片段、 装配所扩增的 DNA片段 以及 PCR扩增全长突变基因。 也可合成全长突变基因， 然后将该全长突变基因克 隆到适当的载体上并转化适当的宿主细胞， 经培养筛选出具有肌酸激酶活性的 阳性克隆。 最后从阳性克隆中提取质粒 DNA， 进行 DNA序列测定分析， 以确定引 入的突变。 在本发明肌酸激酶突变体的制备方法中， 可采用任何适当的载体。 例如， 适用的载体包括但不限于原核表达载体， 如 pRSET和 pES21等； 包括但不 限于克隆载体， 如 PUC18/19 和 pBluscript-SK。
[0027] 在本发明制备肌酸激酶突变体的方法中， 所获得的肌酸激酶突变体基因可以在 原核细胞或真核细胞胞内表达， 也可采用本领域已知的任何其它适当方法实现 在原核细胞或真核细胞胞外表达。
[0028] 在本发明制备肌酸激酶突变体的方法中， 所述载体的宿主细胞为原核细胞或真 核细胞。 所述原核细胞包括但不限于大肠杆菌。 所述真核细胞包括但不限于酿 酒酵母和毕赤巴斯德酵母。
[0029] 本发明中所用的术语 "亲本"系指来自 Oryctolagus cuniculus
的肌酸激酶（CKM)， 其核苷酸序列如序列 1所示（参考 GenBank NM_001082239)， 氨基酸序列如序列 2所示（参考 GenBank NP_001075708)。
[0030] 本发明中所用的术语 "参考序列" ， 当其为核苷酸序列时， 系指序列表中的序 列 1， 当其为氨基酸序列时， 是指序列表中的序列 2。 在将参考序列和突变的肌 酸激酶序列进行排序比较时， 可以手工进行， 也可以用计算机进行 （目前有许 多可供利用的计算机软件， 例如 CLUSTALW程序等） 。
[0031] 本发明中所用的术语 "肌酸激酶突变体"是指这样一种以序列表中序列 2所示 氨基酸序列为参考序列， 存在选自第 100位、 第 121位和第 33位的至少一个突变， 并且以肌酸和三磷酸腺苷 (ATP)为底物生产磷酸肌酸时其具有比亲本高出至少 50 %的肌酸激酶催化活性的酶。 因此， 在本发明中， 所述肌酸激酶突变体的变体， 包括对序列 2所示氨基酸序列中除第 100位、 第 121位和第 33位外的其它位点的保 守取代形式、 增加或缺失一个或几个氨基酸的形式、 氨基端截断的形式、 羧基 端截断的形式、 以及序列 2的部分或全部串联重复形式， 也包括在本发明的范围 内。
[0032] 在发明中所用的氨基酸三字母或单字母表达方式， 采用 IUPAC规定的氨基酸代 码（Eur. J. Biochem. , 138 : 9-37， 1984)。
[0033] 以下用具体的实施例对本发明制备的突变体及其性能进行说明。 下列实施例仅 用于说明本发明而不应视为限定本发明的范围。 实施例中未注明具体条件者， 按常规条件或制造商建议的条件进行。
[0034] 实施例 1 : 肌酸激酶编码基因的扩增与克隆
[0035] 根据基因库（GenBank匪_001082239)基因序列设计引物 ckm_F禾 Pckm-R (见表 1) 。 用弓 I物对 ckm- F禾口 ckm- R从 Oryctolagus cuniculus cDNA文库中扩增肌酸激酶 编码基因。
[0036] 扩增条件为： 20 mM Tris-HCl (pH 8. 8) , 10 mM KC1 , 10 mM (NH4) 2S04， 2 mM MgS04， 0. 1% Triton X-100, 50 mM dATP, 50 mM dTTP, 50 mM dCTP, 50 mM dGTP， 400 nM弓 |物 ckm- F， 400 nM弓 |物 ckm_R， lOOng cDNA, 1. 0 U Pfu DNA 聚合酶 (Promega, USA)， 再用无菌水调反应体积至 50 ml。
[0037] PCR扩增反应程序为： 95 °C 3分钟， 35圈循环： 95 °C 50秒、 50 °C 30秒和 72 °C 1分钟， 最后 72°C 10分钟。 扩增的产物经限制性内切酶 Ndel和 BamHI酶切后与经 同样限制性内切酶 Ndel和 BamHI酶切的载体 pRSET-A (源自 Invitrogen, USA)连接 ， 得质粒 pRSET-ckm。 经 DNA测序， 确定该被克隆的肌酸激酶的核苷酸序列， 具 体示于序列表中序列 1， 相应的氨基酸序列为序列表中的序列 2。
[0038] 表 1
Figure imgf000008_0001
[0039] 实施例 2: 肌酸激酶位点 100的定点突变
[0040] 具体过程如下：
[0041] 为了将亲本氨基酸序列中第 100位点的 Phe (F)突变为 Tyr (Υ)获得突变体 F100Y, 以质粒 pRSET-ckm (见实施例 1)为模板， 设计引物对 100YF和 100YR (见表 1)。
[0042] 用引物对 ckm-F和 100YR, 扩增 F-YR片段， 用引物对 100YF和 ckm_R， 扩增 YF-R片 段。 扩增反应条件为： 20 mM Tris-HCl (pH 8. 8)， 10 mM KC1， 10 mM
(NH4) 2S04, 2 mM MgS04， 0. 1% Triton X- 100， 50 mM dATP, 50 mM dTTP, 50 mM dCTP, 50 mM dGTP， 1. 5 U Pfu DNA聚合酶 （Promega, USA)， 20 ng pRSET-ckm, 以及 400 nM引物 ckm_F和 400 nM引物 100YR (或者， 400
nM引物 100YF和 400 nM引物 ckm-R) ， 用无菌水调反应体积至 50微升。 PCR扩增反 应程序为： 95 °C 3分钟， 35圈循环： 95 °C 50秒、 52 °C 30秒和 72 °C 3分钟， 最 后 72°C 5分钟。 经 1%琼脂糖胶电泳分离并用商业试剂盒回收， 分别得到 F-YR 片段和 YF-R片段。 然后扩增全长基因。 扩增反应条件为： 20 mM Tris-HCl (pH 8. 8)， 10 mM KC1， 10 mM (NH4) 2S04， 2 mM MgS04， 0. 1% Triton X- 100， 50 mM dATP, 50 mM dTTP, 50 mM dCTP, 50 mM dGTP, 400 nM引物 ckm_F和 400 nM ckm-R, 1. 5 U Pfu DNA聚合酶， 20 ng F-YR片段与 20 ng YF-R片段， 用无菌水 调反应体积至 50微升。 PCR扩增反应程序为： 95°C 3分钟， 35圈循环： 95°C 50 秒、 52°C 30秒和 72°C 3分钟， 最后 72°C 5分钟。 经 1%琼脂糖胶电泳分离并用 商业试剂盒回收， 得到全长突变基因 F100Y。 将 F100Y片段回收并经酶切再回收 后与载体 pRSET-A连接（参考实施例 1)， 得质粒 pRSET-F100Y。 将质粒 pRSET-FlOO Y转入感受态细菌细胞 E. col i BL21 o 经 DNA测序确定引入的点突变无误。 F100Y 的氨基酸序列见序列表中的序列 3。 [0043] 实施例 3: 对肌酸激酶突变体位点 121的定点突变
[0044] 为了将亲本氨基酸序列中第 121位点的 Leu (L)突变为 Asp (D)获得突变体 L121D, 以质粒 pRSET-ckm (见实施例 1)为模板， 设计引物对 121DF和 121DR (见表 1)。
[0045] 用引物对 ckm-F和 121DR, 扩增 F-DR片段， 引物对 121DF和 ckm_R， 扩增 DF-R片段 。 引物 ckm-F和 ckm-R的具体序列， 见表 1。 扩增反应条件为： 20 mM Tris-HCl (pH 8. 8)， 10 mM KC1， 10 mM (NH4) 2S04， 2 mM MgS04， 0. 1% Triton
X-100, 50 mM dATP, 50 mM dTTP, 50 mM dCTP, 50 mM dGTP， 400
nM弓 I物 ckm-F禾卩 400 nM引物 121DR，或 400 nM引物 121DF禾口 400 nM引物
ckm-R, 1. 5 U Pfu DNA聚合酶 （Promega, USA)， 20 ng pRSET-ckm, 用无菌水 调反应体积至 50微升。 PCR扩增反应程序为： 95°C 3分钟， 35圈循环： 95°C 50 秒、 52°C 30秒和 72°C 3分钟， 最后 72°C 5分钟。 经 1%琼脂糖胶电泳分离并用 商业试剂盒回收， 分别得到 F-DR片段和 DF-R片段。 然后扩增全长基因。 扩增反 应条件为： 20 mM Tris-HCl (pH 8. 8)， 10 mM KC1， 10 mM (NH4) 2S04， 2 mM MgS04， 0. 1% Triton X-100, 50 mM dATP, 50 mM dTTP, 50 mM dCTP, 50 mM dGTP, 400 nM引物 ckm_F和 400 nM ckm-R, 1. 5 U Pfu DNA聚合酶， 20 ng F-DR片段与 20 ng DF-R片段， 用无菌水调反应体积至 50微升。 PCR扩增反应程 序为： 95 °C 3分钟， 35圈循环： 95 °C 50秒、 52 °C 30秒和 72 °C 3分钟， 最后 72 V 5分钟。 经 1%
琼脂糖胶电泳分离并用商业试剂盒回收， 得到全长突变基因 L121D。 将 L121D与 载体 pRSET-A连接（参考实施例 1)， 得质粒 pRSET-L121D。 将质粒 pRSET_L121D转 入感受态细菌细胞 E. coli BL21 o 经 DNA测序确定引入的点突变无误。 L121D的 氨基酸序列见序列表中的序列 4。
[0046] 实施例 4: 肌酸激酶位点 33的定点突变
[0047] 为了将亲本氨基酸序列中第 33位点的 Val (V)突变为 Ala (A)获得突变体 V33A, 以 质粒 pRSET-ckm (见实施例 1)为模板， 设计引物对 33AF和 33AR (见表 1)。
[0048] 用引物对 ckm-F和 33AR, 扩增 F-AR片段， 引物对 33AF禾 Pckm-R， 扩增 AF-R片段。
引物 ckm-F和 ckm-R的具体序列， 见表 1。 扩增反应条件为： 20 mM Tris-HCl (pH 8. 8)， 10 mM KC1， 10 mM (NH4) 2S04， 2 mM MgS04， 0. 1% Triton X- 100， 50 mM dATP, 50 mM dTTP, 50 mM dCTP, 50 mM dGTP， 400 nM弓 |物 ckm— F禾卩 400 nM 引物 33AR， 或 400 nM引物 33AF和 400 nM引物 ckm_R， 1. 5 U Pfu DNA聚合酶 (Promega, USA)， 20 ng pRSET-ckm, 用无菌水调反应体积至 50微升。 PCR扩增 反应程序为： 95 °C 3分钟， 35圈循环： 95 °C 50秒、 52 °C 30秒和 72 °C 3分钟， 最后 72°C 5分钟。 经 1%琼脂糖胶电泳分离并用商业试剂盒回收， 分别得到
F-AR片段和 AF-R片段。 然后扩增全长基因。 扩增反应条件为： 20 mM
Tris-HCl (pH 8. 8)， 10 mM KC1， 10 mM (NH4) 2S04， 2 mM MgS04， 0. 1%
Triton X-100, 50 mM dATP, 50 mM dTTP, 50 mM dCTP, 50 mM dGTP, 400 nM 引物 ckm-F和 400 nM ckm- R， 1. 5 U Pfu DNA聚合酶， 20 ng F-AR片段与 20 ng AF-R片段， 用无菌水调反应体积至 50微升。 PCR扩增反应程序为： 95°C 3分钟 ， 35圈循环： 95°C 50秒、 52°C 30秒和 72°C 3分钟， 最后 72°C 5分钟。 经 1% 琼脂糖胶电泳分离并用商业试剂盒回收， 得到全长突变基因 V33A。 将 V33A与载 体 pRSET-A连接， 得质粒 pRSET-V33A。 将质粒 pRSET_V33A转入感受态细菌细胞 E. col i BL21 o 经 DNA测序确定引入的点突变无误。 所得突变体的氨基酸序列见序 列表中的序列 5。
[0049] 实施例 5: 亲本肌酸激酶的提取与纯化
[0050] 具体过程如下：
[0051] 将含肌酸激酶基因的质粒 pRSET-ckm转化感受态细菌细胞 E. col i
BL21 , 在 Luria broth (LB)平板（含 50 mg/L卡那霉素）上 37°C培养 24 小时。 接种 单个克隆于 5 毫升 LB液体培养基（含 50 mg/L卡那霉素）中于 30°C培养 20-24小时 。 离心收集菌体， 并悬浮于 1毫升 lOOmM Tris盐酸缓冲液 (pH 7. 5)中。 然后用超 声波裂解细菌细胞。 离心（10°C， 17, 800 g， 10分钟）并收集上清液， 即为粗提 蛋白（或称粗提物）。
[0052] 图 1中 A显示了重组的亲本肌酸激酶粗提蛋白之聚丙烯酰胺凝胶电泳的结果， 表 明肌酸激酶（目标带大小约为 42kD)在大肠杆菌 BL21中有表达。
[0053] 实施例 6: 肌酸激酶突变体的提取与纯化
[0054] 具体过程如下：
[0055] 将含肌酸激酶基因的质粒 pRSET-FlOOY转化感受态细菌细胞 E. col i BL21， 在 Luria broth (LB)平板（含 50 mg/L卡那霉素）上 37 °C培养 24 小时。 接种 单个克隆于 5 毫升 LB液体培养基（含 50 mg/L卡那霉素）中于 30°C培养 20-24小时 。 离心收集菌体， 并悬浮于 1毫升 lOOmM Tris盐酸缓冲液 (pH 7. 5)中。 然后用超 声波裂解细菌细胞。 离心（10°C， 17, 800 g， 10分钟）并收集上清液， 即为粗提 蛋白（或称粗提物）。
[0056] 图 1中 B显示了重组的肌酸激酶突变体 F100Y粗提蛋白之聚丙烯酰胺凝胶电泳的 结果， 表明肌酸激酶（目标带大小约为 42kD)在大肠杆菌 BL21中有较高的表达水 平。
[0057] 实施例 7: 亲本肌酸激酶活性的测定
[0058] 配制底物溶液： 含 lOOmM的肌酸（Creatine)、 5mM之 ATP、 20mM
之 MgC12、 40mM Poly (P)和 lOOmM Tris盐酸缓冲液， 调 pH至 7. 5。 取底物溶液 400 微升， 然后加入 100微升的亲本肌酸激酶，于 37°C进行 10分钟反应。 离心（10°C， 17, 800 g， 15分钟）并收集上清液。 通过高压液相色谱 (HPLC) 测定所得上清液 中磷酸肌酸的含量。 用 SDS聚丙烯酰胺凝胶电泳测定酶蛋白浓度， 测定结果如表 2所示。 一单位酶比活性定义为在上述条件下每分钟转化一微摩尔肌酸为磷酸肌 酸所需之酶量。 肌酸激酶特异性活力为 3. 5U/mg0
[0059] 实施例 8: 肌酸激酶突变体活性的测定
[0060] 配制底物溶液： 含 lOOmM的肌酸（Creatine)、 5mM之 ATP、 20mM
之 MgC12、 40mM Poly (P)和 lOOmM Tris盐酸缓冲液， 调 pH至 7. 5。 取底物溶液 400 微升， 然后加入 100微升肌酸激酶突变体 F100Y，于 37°C进行 10分钟反应。 离心（1 0°C， 17, 800 g， 15分钟）并收集上清液。 通过高压液相色谱 (HPLC) 测定所得上 清液中磷酸肌酸的含量。 用 SDS聚丙烯酰胺凝胶电泳测定酶蛋白浓度， 测定结果 如表 2所示。 一单位酶比活性定义为在上述条件下每分钟转化一微摩尔肌酸为磷 酸肌酸所需之酶量。 肌酸激酶特异性活力为 7. 4U/mg0
[0061] 表 2 肌酸激酶亲本与突变体酶比活性之差异
[] [表 1]
Figure imgf000012_0001
[0062] 实施例 9: 肌酸激酶固定化
[0063] 取肌酸激酶粗酶， 用洗酶缓冲液 （0. 02M Tris-HCl/O. OOlM EDTA， pH7. 0溶液 ) 稀释至蛋白含量 5-10mg/ml。 将酶稀释液与 PB溶液（2. Omol/L磷酸二氢钾， PH7. 5)等体积混合， 加入固定化酶载体 LX-3000 (10毫克酶 /克载体）， 于摇床（ 转速 lOOrpm)中 25° C反应 20小时。 反应完成后用滤袋过滤， 用洗酶缓冲液清洗 5 -6次， 得到固定化肌酸激酶。
[0064] 实施例 10: 用固定化肌酸激酶制备磷酸肌酸
[0065] 配制底物溶液： 含 lOOmM的肌酸（Creatine)、 5mM之 ATP、 20mM之 MgCl 2
、 40mM Poly (P)和 lOOmM Tris盐酸缓冲液， 调 pH至 7. 5。 取底物溶液 1毫升， 然 后加入 0. 05克固定化肌酸激酶。 于 37°C进行反应 2-20小时。 离心（10°C， 17, 800 g， 15分钟）并收集上清液。 通过高压液相色谱 (HPLC) 测定所得上清液中磷酸肌 酸的含量。 结果， 肌酸转化为磷酸肌酸的转化率超过 85%。
[0066] 应当理解的是， 本发明的应用不限于上述的举例， 对本领域普通技术人员来说 ， 可以根据上述说明加以改进或变换， 所有这些改进和变换都应属于本发明所 附权利要求的保护范围。

Claims

权利要求书
[权利要求 1] 一种肌酸激酶突变体， 其特征在于， 以序列表的序列 2为参考序列， 其具有选自第 100位、 第 121位和第 33位的至少一个突变， 并且以三磷 酸腺苷和肌酸为底物时其具有比亲本高出至少 50%的肌酸激酶催化活 性。
[权利要求 2] 根据权利要求 1所述的肌酸激酶突变体， 其特征在于， 其中第 100位的 苯丙氨酸突变为酪氨酸。
[权利要求 3] 根据权利要求 1所述的肌酸激酶突变体， 其特征在于， 其中第 121位的 亮氨酸突变为天冬氨酸。
[权利要求 4] 根据权利要求 1所述的肌酸激酶突变体， 其特征在于， 其中第 33位的 缬氨酸突变为丙氨酸。
[权利要求 5] 根据权利要求 1所述的肌酸激酶突变体， 其特征在于， 其具有所附序 列表中 SEQ ID NO. : 3至 SEQ ID NO.： 5之一所示的氨基酸序列。
[权利要求 6] 一种基因， 其特征在于， 其含有编码权利要求 1-5任一项所述的肌酸 激酶突变体的核苷酸序列。
[权利要求 7] 一种如权利要求 1-5任一项所述的肌酸激酶突变体的应用， 其特征在 于， 将其应用于以三磷酸腺苷和肌酸为底物制备磷酸肌酸的过程中。
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