WO1999027115A1 - Phosphoesterase alcaline calorifuge et son expression - Google Patents

Phosphoesterase alcaline calorifuge et son expression Download PDF

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Publication number
WO1999027115A1
WO1999027115A1 PCT/CN1998/000272 CN9800272W WO9927115A1 WO 1999027115 A1 WO1999027115 A1 WO 1999027115A1 CN 9800272 W CN9800272 W CN 9800272W WO 9927115 A1 WO9927115 A1 WO 9927115A1
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ala
leu
gcc
gly
val
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PCT/CN1998/000272
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English (en)
French (fr)
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Xiaoyu Sheng
Yumin Mao
Youzhong Yuan
Chaoneng Ji
Zongxiang Zhou
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Fudan University
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Priority to US09/530,851 priority Critical patent/US6649390B1/en
Priority to AU11402/99A priority patent/AU1140299A/en
Priority to EP98954109A priority patent/EP1038964A4/en
Publication of WO1999027115A1 publication Critical patent/WO1999027115A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)

Definitions

  • the present invention belongs to the technical field of bioengineering, and relates to a heat-resistant alkaline phosphatase.
  • Alkaline phosphatase is an important enzyme that is widely present in various organisms and is involved in cellular phosphorus metabolism.
  • Alkaline phosphatase is a non-specific phosphodiesterase that produces inorganic phosphorus and alcohol by forming an intermediate of phosphoserine.
  • Alkaline phosphatase amino acids have been obtained from a variety of prokaryotes and eukaryotes. Sequence and corresponding genes, such as E. coli, Bacillus subtilis, yeast, bovine intestine, human placenta, etc.
  • Alkaline phosphatase is an important tool for molecular biology research, such as dephosphorization of DNA or RA fragment ends in gene cloning, enzyme-linked reagents in immunological research, and nucleic acid markers for nucleic acids. Hybridization or detection of polymerase chain reaction products, etc.
  • Nucleic acid hybridization uses a piece of labeled DNA or RNA as a probe and detects a complementary nucleic acid sequence. It is one of the most widely used technologies in molecular biology.
  • the label of a nucleic acid probe is usually 32 P or 35 S equivalent isotope, although highly sensitive, but due to short half-life, harm to the human body during operation and the tedious handling of isotope waste, it is significantly restricted in routine applications in biology and medicine and commercial sales of kits. Nearly a dozen Over the years, extensive research has been performed on labeling nucleic acid probes with non-isotopic substances (Mattews J. Anal Biochem. 1988, 169: 1-25).
  • Indirect labeling mainly includes biological Haptens such as digoxigenin and digoxin; and direct labeling are mainly enzymes and luciferin.
  • Alkaline phosphatase is the most widely used enzyme in both indirect and direct labeling. Alkaline phosphate has been used in the 1980s. Reports on the direct labeling of nucleic acids by esterases (Jablonski E: Nucleic Acids Res 1986, 14: 6115-6128).
  • the alkaline phosphatase used is mainly bovine small intestine alkaline phosphatase or E. coli alkaline phosphatase.
  • the main disadvantage of enzymes is that they are not heat resistant and are not suitable for hybridization at higher temperatures, and higher temperature hybridization is conducive to reducing the background of hybridization and increasing specificity; nor can it tolerate strong hybridization such as high concentration SDS Elution conditions. Due to their low heat resistance, these alkaline phosphatase-labeled oligonucleotides cannot be used as primers for polymerase chain reaction (PCR).
  • thermophilic bacteria are a kind of microorganisms that grow in the above environment.
  • the enzymes of thermophilic bacteria are mostly thermostable enzymes, which have high application value, such as the widespread application of thermostable DNA polymerase in PCR technology. But Prior to the present invention, no reports and patents derived from thermophilic alkaline phosphatase.
  • the object of the present invention is to provide an alkaline phosphatase with high heat resistance and wider application.
  • the "heat-resistant alkaline phosphatase” (abbreviated as FD-TAP) referred to in the present invention refers to alkaline phosphatase having the following properties or characteristics:
  • the optimum reaction temperature is above 50'C, and 70 ' When the water bath of C is incubated for 30 minutes, more than 70% of the enzyme activity is still observed.
  • the above properties or characteristics are measured in the most suitable storage and reaction system. As the reaction system or conditions change, The nature or characteristics may fluctuate.
  • thermostable alkaline phosphatase which is homologous or substantially homologous to the amino acid sequence shown in Table 1,
  • N-terminus of the amino acid sequence of 496 Pro Glu Asp Val Trp Ala is marked with a horizontal line and a signal peptide consisting of 26 amino acid residues.
  • the invention also provides DNA fragments that are homologous or substantially homologous to the nucleotide sequences encoding the enzymes of the invention as shown in Table 2.
  • DNA fragment includes single- or double-stranded DNA in the present invention.
  • “Homologous” in the present invention means according to the specific content: (1) a DNA fragment and another DNA fragment have exactly the same nucleotide sequence; (2)-a protein has the same amino acid sequence as another protein.
  • Base homology means (1) that one DNA fragment has enough nucleotide sequences with another fragment, so that the proteins translated by the two have similar properties or characteristics; (2) one protein is sufficient with another protein The same amino acid sequence makes both of them show similar properties and characteristics.
  • thermostable alkaline phosphatase and its DNA fragments such as prokaryotes, yeasts, and mammals.
  • prokaryotes especially thermophiles, such as commercially available Thermomusus bacteria are preferred. Wait,
  • the DNA fragment encoding the thermostable alkaline phosphatase can be partially deleted by genetic engineering.
  • the DNA fragment can be continuously deleted from the 5 'end to the 3' end or from the 3 'end to the 5' end, or from two The end to the middle is continuously deleted, or the middle part can be deleted, and then the DNA at both ends can be connected.
  • the shortest DNA fragment can be as short as 60 bases.
  • the polypeptide encoded by the deleted DNA fragment generally still retains heat resistance Properties and characteristics of alkaline phosphatase.
  • the present invention also provides a recombinant vector containing one or more copies of the DNA fragment (or deleted DNA fragment) of the present invention, which vector is capable of expressing the thermostable alkaline phosphatase of the present invention in a host.
  • the vector of the present invention may be a eukaryotic vector or a prokaryotic vector, but it is preferably a prokaryotic vector, so as to facilitate the proliferation of prokaryotic organisms.
  • Prokaryotic vectors can be lambda phages (such as ⁇ ⁇ 1 1, 7S> ash, ZapII, etc.), or plasmids (such as pBR322, pUC series, pBluescript, etc.), but preferably plasmids, these vectors can be obtained from commercial sources.
  • the present invention also provides a microorganism transformed by the recombinant vector of the present invention, of which the preferred is Gram-negative bacilli, especially E. coli.
  • the recombinant vector of the present invention can be obtained by the following steps:
  • step (3) screening the gene library of step (2) by an appropriate method
  • Prokaryotes can be treated with lysozyme and proteinase K can be added to isolate chromosomal DNA.
  • DNA can be digested with suitable restriction enzymes by methods familiar to those skilled in the field of molecular biology.
  • the digested DNA is ligated into a suitable cloning vector, and a suitable organism is transformed with a recombinant vector to generate a gene library.
  • suitable restriction enzymes for specific methods, please refer to the genetic engineering manual (Sambrook J, et al. In: Molecular Cloning, A laboratory Manual. 2 ed, CSH Press, 1989).
  • thermostable alkaline phosphatase is simple and convenient, and the thermostable property of the enzyme is used. Positive colonies expressing thermostable alkaline phosphatase can be selected on in situ colonies.
  • Positive clone insert DNA fragments can be sequenced in both directions using Sanger dideoxy termination
  • the invention also includes a method for producing a thermostable alkaline phosphatase having homology or substantially homology to the amino acid sequence in FIG. 1:
  • the recombinant thermotolerant alkaline phosphatase of the present invention can be expressed in a transformed host under the control of any suitable promoter and translation element.
  • the host includes prokaryotes, yeast, mammalian cells, insects and plants, preferably prokaryotes Organisms, especially E. coli.
  • the choice of vector varies depending on the recipient.
  • plasmids are used as expression vectors in E. coli, such as pJLA503 (Lehauder B, et al. Gene, 1987; 53: 279-283), pET series (Stratagene Co.) and the like.
  • Escherichia coli is a multi-media rich medium, such as 2 ⁇ ⁇ etc. depending on the kind of carrier, temperature or chemical methods can be employed (e.g., IPTG, etc.) to induce expression of the enzyme protein.
  • the heat-resistant alkaline phosphatase can be isolated and purified from the cultured cells or from the culture medium.
  • the expressed enzyme protein exists in the cells. After the cells are centrifuged, they can be broken by ultrasound, lysozyme, and freeze-thaw methods. Centrifuge or filter and take the supernatant to obtain the crude enzyme product; if the enzyme protein is secreted into the culture medium, the cells can be centrifuged to remove the enzyme, and then the enzyme can be purified from the supernatant.
  • There are various methods for purifying the enzyme protein such as salting out, ultrafiltration Filtration, dialysis, ion-exchange chromatography, HPLC, etc.
  • the enzyme product is kept at a higher temperature (such as 60'C) for a certain period of time to effectively remove impurities, which greatly simplifies the purification process. And convenient.
  • Thermostable alkaline phosphatase can be used for the labeling of nucleic acids, proteins or other biological macromolecules, and can also be used for the dephosphorization treatment of the ends of DNA or RNA molecules in gene cloning.
  • the most important use is the direct Labeling, labeled nucleic acids or oligonucleotides have three main uses: First, as a probe for nucleic acid hybridization and footprint analysis, including Southern blot, Northern blot, Slot blot, dot blot, Southern-western blot, in situ hybridization The second is used as a primer for nucleic acid in vitro amplification, and the third is used for DNA sequence analysis.
  • Enzyme protein and nucleic acid or oligonucleotide are mainly linked by chemical or physical methods such as covalent cross-linking, which can be through cross-linking.
  • the arm connects the terminal group of the nucleic acid or oligonucleotide with the amino group or thiol group of the enzyme protein (Ruth et ah DNA 1985; 4:93).
  • the detection method can be chemical, physical or biological methods.
  • the solid Phase hybridization differs from liquid phase hybridization.
  • the former uses the chromogenic method with BCIP / NBT as the substrate or the chemiluminescence method with AMPPD as the substrate (Schaap A, et al. Clin. Chem. 1989, 35: 1863-1864) As well
  • the latter uses p-nitrophenol (pNPP) as a substrate, which can be quantitatively determined.
  • pNPP p-nitrophenol
  • Figure 1 The nucleotide sequence of the thermostable alkaline phosphatase gene and the deduced amino acid sequence.
  • the amino acid sequence is marked below the DNA sequence (the amino acid residue is represented by three letters), and the N-terminus of the amino acid sequence is marked with a horizontal line. A signal peptide with 26 amino acid residues is shown.
  • Figure 2 Schematic representation of the highly expressed plasmid pTAP503 cloned with the FD-TAP gene.
  • FIG 3 Heat resistance of FD-TAP.
  • the measurement method is to incubate the enzyme solution in a 95'C water bath for different times, and then measure the enzyme activity.
  • Figure 4 Optimum temperature of FD-TAP.
  • TE 1 Ommol L Tris-HCl, lmmol / L EDTA, pH8.0
  • TH 0.3% peptone, 0.3% yeast extract, 0.2% NaCl, pH 7.0
  • LB 1% peptone, 0.5 ° /. Yeast extract, 1% NaCl, pH7.0
  • FD-TAP Thermostable alkaline phosphatase from 77ie 7m «sp. FD3041.
  • Example 1 sp.FD3041 Isolate chromosomal DNA
  • 3 ⁇ 4er / m «sp. FD3041 (available from Fuhua Industrial Co., Ltd., Shanghai, China) was cultured at 70'C in 200 ml of TH liquid medium.
  • the bacterial solution was collected by centrifugation, suspended in 12 ml of TE buffer, and added with 1 ml of TE buffer containing 10 mg / ml of lysozyme, and was incubated in a 37'C water bath for 2 hours, and added with 1.5 ml of 10 ° /.
  • the above gene library was screened by the colony in situ alkaline phosphatase coloring method, as follows: The colonies were transferred to 3MM filter paper, and immersed in a bactericidal buffer solution (lmol / L diethanolamine, 1% SDS) at 85 ⁇ Incubate in water bath for 10min, then immerse in reaction buffer (6mmol / L pNPP, 1mol / L diethanolamine, 1 ° /. SDS) in 70: Incubate in water bath for 10min, the color of colony turns yellow is positive. By screening, get 5 Positive clones. Right One of the clones (PTAP362) was subjected to a physical map and the TAP activity of the partially deleted plasmid was determined to locate the FD-TAP gene on a 2 kb DNA fragment.
  • a bactericidal buffer solution lmol / L diethanolamine, 1% SDS
  • reaction buffer 6mmol / L pNPP, 1mol / L diethanolamine, 1
  • the DNA sequence was determined by Sanger dideoxy chain termination method, and the obtained nucleotide sequence was 2030bp ( Figure 1).
  • the computer analysis showed that the gene length of FD-TAP was 1506bp, (3 +% was 68.2%, the third base of the codon 0 +% was 92.7%, which was in line with the gene characteristics of Thermus genus.
  • the gene encodes a gene consisting of 501
  • the original enzyme protein consisting of amino acid residues has a signal peptide sequence of 26 amino acid residues at the N-terminus, and the thermogenic enzyme protein molecule consists of 475 amino acid residues.
  • the DNA sequence of the FD-TAP gene and the protein encoding protein The amino acid sequence is shown in Figure 1.
  • Example 3 Subcloning and high expression of the FD-TAP gene:
  • Primers were designed at the start codon and the stop codon of the FD-TAP gene. The 5 'ends of the primers have Ndel and BamHI digestion sites, respectively.
  • the PCR products were amplified from the plasmid pTAP118B to obtain mature primers.
  • FD-TAP gene sequence The gene was cloned into the high expression vector pJLA503 after enzymatic digestion, and transformed with the phoA gene-deficient strain Mph44.
  • the recombinant transformants were screened by colony in situ coloration method on LB plates containing ampicillin. Results 50% of the transformants were positive clones expressing FD-TAP.
  • DNA sequence analysis of the recombinant plasmid (denoted as pTAP503, see Figure 2) contained in one of the clones showed that no mutation occurred in the gene.
  • E. coli Mph44 (pTAP503F) was induced at 42'C for 10 hours after 30'C liquid culture. SDS-PAGE showed that there was an enzyme expression product at about 53kDa. About 10% of the total protein.
  • Example 4 Recombinant FD- Isolation and purification of TAP:
  • the bacterial solution is centrifuged at 15,000 rpm for 15 minutes, the precipitate is discarded and collected.
  • PEI concentration 0.04% PEI concentration 0.04%
  • centrifuge at 15,000 rpm for 15 minutes discard the precipitate, and collect the supernatant.
  • NaCl NaCl
  • heat-denatured in a 70 'C water bath for 30 minutes and centrifuged at 5,000 rpm for 15 minutes to remove the heat-resistant heterologous proteins from the total cell protein and collect the supernatant.
  • reaction system 6 mmol / L, p-nitrophenol, 1 mol / L diethanolamine, pH l 1.6
  • reaction system 6 mmol / L, p-nitrophenol, 1 mol / L diethanolamine, pH l 1.6
  • 70'C water bath for 10 minutes
  • the reaction was stopped with acetic acid solution, and the absorbance of the product at OD405 was measured on UV260.
  • Definition of enzyme unit At 70'C, pHl 1.6, the amount of enzyme that catalyzes the production of lpmol / L of p-nitrophenol per minute is defined as one enzyme unit.
  • Enzyme unit A405x 2 / (18.8x10). Of which 2 Is the total reaction volume, 10 is the reaction time, and the molar extinction coefficient of p-nitrophenol at 405 nm is 18.8 0 6 .
  • thermostable alkaline phosphatase (ii) Title of invention: a thermostable alkaline phosphatase and its expression
  • T r Asp Gly Phe Ser Trp Glu Asp Tyr Ala lie Ala Gin Ala Tyr
  • Pro Asp Arg Pro Asn lie Gly Trp Ser Ser Gly Gin His Thr Ala

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Description

一种耐热碱性磷酸酯酶及其表达 本发明属生物工程技术领域， 涉及一种耐热碱性磷酸酯酶.
碱性磷酸酯酶是广泛存在于各种生物体内、 参与细胞磷代谢的重要酶类。 碱 性磷酸酯酶是一种非特异性的磷酸单酯酶， 通过形成磷酸丝氨酸的中间体， 产生 无机磷和醇. 已经从多种原核生物和真核生物内获得了碱性磷酸酯酶的氨基酸序 列以及相应的基因， 如大肠杆菌， 枯草杆菌， 酵母， 牛小肠， 人胎盘等（ Bradshaw RA: PNAS 1981,78:3473-3477; Kam W: PNAS 1985, 82:8715-8719; Milan JL: J Bio Chem 1986, 261:3112-3115; Hulett FM: J Biol Chem. 1991, 266: 1077-84 ) .
碱性磷酸酯酶是分子生物学研究的重要工具酶， 如在基因克隆中用于 DNA 或 R A片段末端的去磷处理、 在免疫学研究中用作酶联试剂、 作为核酸标记物用 于核酸杂交或检测聚合酶链反应的产物等.
核酸杂交是以一段标记的 DNA或 RNA做为探针， 检测与其互补的核酸序列 的一种技术， 是分子生物学中最为广泛应用的技术之一. 核酸探针的标记物通常 是 32P或 35S等同位素， 虽然敏感性高， 但由于半衰期短、 操作中对人体的危害及 同位素废物的处理繁琐等缺点， 在生物学和医学常规应用和商业销售试剂盒方面 受到明显限制. 近十多年来， 应用非同位素物质标记核酸探针已进行了广泛的研 究 ( Mattews J . Anal Biochem. 1988,169:1-25 ) .
目前常用的标记物包括酶，荧光素，生物素，地高辛等（欧洲专利 EP304934 ). 根据标记物在杂交后是否能直接检测而分为直接标记和间接标记两种. 间接标记 主要有生物素和地高辛等半抗原； 而直接标记主要有酶和荧光素. 碱性磷酸酯酶 无论是间接标记还是直接标记中都是最为广泛应用的酶类. 在八十年代已有用碱 性磷酸酯酶直接标记核酸的报道 （ Jablonski E: Nucleic Acids Res 1986, 14:6115-6128 ) , 所用的碱性磷酸酯酶主要是牛小肠碱性磷酸酯酶或大肠杆菌的 碱性磷酸酯酶， 这些酶的主要缺点是耐热性不高， 不适用于在较高温度下进行杂 交， 而较高的温度杂交有利于减少杂交背景、 增加特异性； 也不能耐受如高浓度 SDS等强烈的杂交洗脱条件. 由于耐热性不高， 这些碱性磷酸酯酶直接标记的寡 核苷酸也不能作为聚合酶链反应（PCR) 的引物.
嗜热菌是一类在 以上的环境中生长的微生物.嗜热菌所具有的酶多为耐 热酶， 有很高的应用价值， 如耐热 DNA聚合酶在 PCR技术中的广泛应用. 但在本 发明之前， 未见来源于嗜热菌碱性磷酸酯酶的报道和专利.
本发明的目的是提供一种耐热性高、 用途更为广泛的碱性磷酸酯酶. 本发明所说的 "耐热碱性磷酸酯酶"（简记 FD-TAP)是指具有下述性质或特征 的碱性磷酸酯酶： 最适反应温度在 50'C以上， 并且在 70'C的水浴保温 30分钟时仍 有 70%以上的酶活. 对同一种酶而言， 上述性质或特征是在最适的保存和反应体 系中测得的， 随着反应体系或条件的改变， 该性质或特征可能有所波动。
本发明提供了一种耐热碱性磷酸酯酶， 该酶与表 1所示的氨基酸序列同源或 基本同源，
表 1.耐热碱性磷酸酯酶的氨基酸序列
1 Met Lys Arg Arg Asp He Leu Lys Gly Gly Leu Ala Ala Gly Ala
16 Leu Ala Leu Leu Pro Arg Gly His Thr Gin Gl.y Ala Leu Gin Asn
31 Gin Pro Ser Leu Gly Arg Arg T r Arg Asn Leu lie Val Phe Val
46 Tyr Asp Gly Phe Ser Trp Glu Asp T r Ala He Ala Gin Ala Tyr
61 Ala Arg Arg Arg Gin Gly Arg Val Leu Ala Leu Glu Arg Leu Leu
76 Ala Arg Tyr Pro Asn Gly Leu He Asn Thr T r Ser Leu Thr Ser
91 Tyr Val Thr Glu Ser Ser Ala Ala Gly Asn Ala Phe Ser Cys Gly
106 Val Lys Thr Val Asn Gly Gly Leu Ala He His Ala Asp Gly Thr
121 Pro Leu Lys Pro Phe Phe Ala Ala Ala Lys Glu Ala Gly Lys Ala
136 Val Gly Leu Val Thr Thr Thr Thr Val Thr His Ala Thr Pro Ala
151 Ser Phe Val Val Ser Asn Pro Asp Arg Asn Ala Glu Glu Arg lie
166 Ala Glu Gin T r Leu Glu Phe Gly Ala Glu Val Tyr Leu Gly Gly
181 Gly Asp Arg Phe Phe Asn Pro Ala Arg Arg Lys Asp Gly Lys Asp
196 Leu Tyr Ala Ala Phe Ala Ala Lys Gly Tyr Gly Val Val Arg Thr
211 Pro Glu Glu Leu Ala Arg Ser Asn Ala Thr Arg Leu Leu Gly Val
226 Phe Ala Asp Gly His Val Pro Tyr Glu He Asp Arg Arg Phe Gin
241 Gly Leu Gly Val Pro Ser Leu Lys Glu Met Val Gin Ala Ala Leu
256 Pro Arg Leu Ala Ala His Arg Gly Gly Phe Val Leu Gin Val Glu
271 Ala Gly Arg He Asp His Ala Asn His Leu Asn Asp Ala Gly Ala
286 Thr Leu Trp Asp Val Leu Ala Ala Asp Glu Val Leu Glu Leu Leu
301 Thr Ala Phe Val Asp Arg Asn Pro Asp Thr Leu Leu Leu Val Val
316 Ser Asp His Ala Thr Gly Val Gly Ala Leu Ty「 Gly Ala Gly Arg
331 Ser Tyr Leu Glu Ser Ser Val Gly lie Asp Leu Leu Gly Ala Gin
346 Lys Ala Ser Phe Glu T r Met Arg Arg Val Leu Gly Ser Ala Pro
361 Asp Ala Ala Gin Val Lys Glu Ala Tyr Gin Thr Leu Lys Gly Val 376 Ser Leu Thr Asp Glu Glu Ala Gin Met Val Val Arg Ala He Arg
391 Glu Arg Val T r Trp Pro Asp Ala Val Arg Gin Gly lie Gin Pro
406 Glu Asn Thr Met Ala Trp Ala Met Val Gin Lys Asn Ala Ser Lys
421 Pro Asp Arg Pro Asn He Gly Trp Ser Ser Gly Gin His Thr Ala
436 Ser Pro Val lie Leu Leu Leu Tyr Gly Gin Gly Leu Arg Phe Val
451 Gin Leu Gly Leu Val Asp Asn Thr His Val Phe Arg Leu Met Gly
466 Glu Ala Leu Asn Leu Arg T r Gin Asn Pro Val Met Ser Glu Glu
481 Glu Ala Leu Glu He Leu Lys Ala Arg Pro Gin Gly Met Arg His
496 Pro Glu Asp Val Trp Ala 氨基酸序列的 N端用横线标出了由 26个氨基酸残基组成的信号肽.
本发明也提供了与表 2所示的编码本发明酶的核苷酸序列同源或基本同源的 DNA片段.
表 2. 耐热碱性磷酸酯酶基因的核苷酸序列
1 ATG AAG CGA AGG GAC ATC CTG AAA GGT GGC CTG GCT GCG GGG GCC
46 CTG GCC CTC CTG CCC CGG GGC CAT ACC CAG GGG GCT CTG CAG AAC
91 CAG CCT TCC丌 G GGA AGG CGG TAC CGC AAC CTC ATC GTC丌 C GTC
136 TAC GAC GGG TTT TCC TGG GAG GAC TAC GCC ATC GCC CAG GCC TAC
181 GCC CGG AGG CGG CAG GGC CGG G丌 CTC GCC CTG GAG CGC CTC CTC
226 GCC CGC TAC CCC AAC GGG CTC ATC AAC ACC TAC AGC CTC ACC AGC
271 TAC GTC ACC GAG TCC AGC GCC GCG GGG AAC GCC TTC TCC TGC GGG
316 GTG AAG ACG GTG AAC GGG GGG CTC GCC ATC CAC GCC GAC GGG ACC
361 CCC CTC AAG CCC TTC丌 C GCC GCG GCC AAG GAG GCG GGG AAG GCC
406 GTG GGG CTC GTG ACC ACC ACC ACC GTC ACC CAC GCC ACC CCG GCG
451 AGC TTC GTG GTG TCC AAT CCC GAC CGG AAC GCC GAG GAG AGG ATC
496 GCC GAG CAG TAC CTG GAG TTC GGG GCC GAG GTG TAC CTT GGG GGC
541 GGG GAC CGC πτ丌 C AAC CCC GCC AGG CGC AAG GAC GGG AAG GAC
586 CTC TAC GCC GCC TTC GCC GCC AAG GGG TAC GGG GTG GTG CGC ACC
631 CCC GAG GAG CTC GCC CGT TCC AAC GCC ACC CGG CTC CTG GGC GTC
676 丌 C GCC GAC GGC CAC GTG CCC TAC GAG ATT GAC CGC CGC TTC CAG
721 GGC C丌 GGG GTG CCG AGC CTC AAG GAA ATG GTC CAG GCC GCT TTG
766 CCC CGG CTT GCC GCC CAC CGC GGG GGC TTC GTC CTT CAG GTG GAA 811 GCG GGG CGG ATT GAC CAC GCC AAC CAT TTG AAC GAC GCC GGG GCC
856 ACC CTT TGG GAC GTG CTG GCG GCG GAC GAG GTC 丌 G GAG C丌 CTC
901 ACC GCC TTC GTG GAC CGG AAC CCG GAC ACC CTC CTC CTC GTG GTC
946 TCG GAC CAC GCC ACC GGG GTG GGG GCC CTC TAC GGG GCG GGC CGG
991 AGC TAC CTG GAG AGC TCC GTG GGC ATT GAC CTC CTG GGG GCG CAA
1036 AAG GCC AGC TTT GAG TAC ATG CGC CGC GTC TTG GGC TCG GCC CCC
1081 GAT GCT GCC CAG GTG AAG GAG GCC TAC CAG ACC CTG AAG GGG GTC
1126 TCC CTC ACG GAC GAG GAG GCG CAG ATG GTG GTC CGG GCC ATC CGC
1171 GAG CGG GTC TAC TGG CCT GAT GCC GTG CGC CAG GGC ATC CAG CCC
1216 GAA AAC ACC ATG GCC TGG GCC ATG GTG CAG AAG AAC GCC AGC AAG
1261 CCC GAC CGG CCC AAC ATC GGC TGG AGC TCT GGG CAG CAC ACG GCG
1306 AGC CCC GTC ATC CTC CTC CTC TAC GGC CAG GGC CTG CGC TTC GTC
1351 CAG CTT GGC CTG GTG GAC AAC ACC CAC GTG TTC CGC CTG ATG GGC
1396 GAG GCC CTG AAC CTC CGC TAC CAG AAC CCG GTG ATG AGC GAG GAG
1441 GAG GCC CTG GAG ATC CTC AAG GCC AGG CCC CAG GGG ATG CGC CAC
1486 CCC GAG GAC GTC TGG GCC TAA 上述短语 "DNA片段" 在本发明中包括单链或双链的脱氧核糖核酸.
"同源" 在本发明中根据具体内容表示： （1 )一个 DNA片段与另一个 DNA 片段有完全相同的核苷酸序列； （2 )—个蛋白质与另外一个蛋白质有完全相同 的氨基酸序列.
"基本同源" 表示（ 1 )一个 DNA片段与另外一个片段有足够相同的核苷酸 序列， 使得两者翻译的蛋白质有相似的性质或特征； （2 )—个蛋白质与另一个 蛋白质有足够相同的氨基酸序列， 使得两者表现出相似的性质和特征.
各种生物体都可以作为耐热碱性磷酸酯酶及其 DNA片段的来源， 如原核生 物， 酵母和哺乳动物等. 但首选为原核生物， 尤其是嗜热菌， 例如市售的 Thermus thermophilus菌等，
编码耐热碱性磷酸酯酶的 DNA片段可以通过遗传工程的方法使之部分缺 失. 可以从 DNA片段的 5'端向 3'端或从 3'端向 5'端进行连续缺失， 或从两端向中间 连续缺失， 也可以缺失中间部分， 再将两端部分的 DNA连接起来. 缺失后的 DNA 片段最短可仅有 60个碱基.缺失后的 DNA片段编码的多肽一般仍保留有耐热碱性 磷酸酯酶的性质和特征. 本发明也提供了含有本发明 DNA片段 (或缺失后的 DNA片段)的一个或多个 拷贝的重组载体， 该载体能够在宿主中表达本发明的耐热碱性磷酸酯酶.
本发明的载体可以是真核载体， 也可以是原核载体， 但最好是原核载体， 以 便于在原核生物体中增埴。 原核载体可以是 λ噬菌体 (如 λ ί1 1， 7S>ash, ZapII等)， 也可以是质粒（如 pBR322, pUC系列， pBluescript等） ， 但最好是质粒， 这些载体 都可以从商业途径得到.
本发明也提供了一种被本发明重组载体转化的微生物，其中首选的是革兰氏 阴性杆菌， 尤其是大肠杆菌.
本发明的重组载体可通过以下步骤获得：
( 1 )分离原核生物的染色体 DNA, 并用合适的限制性内切酶进行消化，
( 2 )将消化后的 DNA整合到一载体中， 用该重组载体转化合适的宿主， 并 以此方式构建基因文库；
( 3 )用适当的方法筛选步骤（2 ) 的基因文库；
( 4 )对步骤（3 )筛选的阳性克隆进行分析.
可使用溶菌酶处理原核生物， 并加入蛋白酶 K来分离染色体 DNA.
可按分子生物学领域熟练技术人员熟悉的方法， 用合适的限制酶消化 DNA. 将消化后的 DNA连接到合适的克隆载体上，并用重组载体转化合适的生物体以产 生基因文库。 具体操作方法可参见基因工程的操作手册 (Sambrook J, et al. In: Molecular Cloning, A laboratory Manual. 2 ed, CSH Press, 1989).
可用以下的方法从文库中筛选基因： A.寡核苷酸探针杂交; B.用聚合酶链反应 方法; C.用抗体筛选; D.根据酶活性筛选. 其中用核酸或寡核苷酸探针进行菌落的 原位杂交是常用的方法， 但在本发明中， 以酶活性筛选为首选， 因为耐热碱性磷 酸酯酶的活性检测简单方便， 且利用该酶的耐热的性质， 可在原位菌落上筛选表 达耐热碱性磷酸酯酶的阳性克隆.
阳性克隆的插入 DNA片段可采用 Sanger双脱氧终止法进行双向的 DNA测序
(常规的放射性同位素手工测序或用自动测序仪进行自动测序） . 其结果如图 1 所示.
本发明还包括生产具有与图 1中氨基酸序列同源或基本同源的耐热碱性磷酸 酯酶的方法：
( 1 ) 用编码本发明酶的 DNA片段 (或部分缺失后的 DNA片段)或含有该 DNA 片段的重组载体转化合适的宿主；
( 2 )在合适的培养基中培养转化后的宿主； ( 3 )从培养基或细胞中分离、 纯化蛋白质。
可以在任何合适的启动子和翻译元件控制下在转化的宿主中表达本发明的 重组耐热碱性磷酸酯酶. 宿主有原核生物、 酵母、 哺乳动物细胞、 昆虫和植物等, 最好是原核生物， 尤其是大肠杆菌. 载体的选择根据受体的不同而异， 在大肠杆 菌中一般为以质粒为表达载体， 如 pJLA503 (Lehauder B， et al. Gene, 1987;53:279- 283), pET系列 (Stratagene公司产品)等. 大肠杆菌的培养基多为丰富培养基， 如 2 ΧΥΤ等. 根据载体的种类不同， 可采用温度或化学方法（如 IPTG等）诱导酶蛋白 的表达.
可从培养的细胞中或培养基中分离纯化耐热碱性磷酸酯酶.如表达的酶蛋白 存在于细胞中， 细胞经离心后， 可用超声波、 溶菌酶、 冻融法等破菌后，经离心或 过滤，取上清获得酶粗制品；如酶蛋白分泌到培养基中， 可经离心去细胞后，再从 上清液纯化酶. 纯化酶蛋白有多种方法， 如盐析法、 超滤法、 透析法、 离子交换 层析和 HPLC等. 在纯化过程中， 酶制品在较高的温度下 (如 60'C )中保温一定的时 间可有效去除杂蛋白， 使纯化过程大为简化和方便。
耐热碱性磷酸酯酶可用于核酸、 蛋白质或其它生物大分子的标记， 也可用作 基因克隆中 DNA或 RNA分子的末端去磷处理.其中最主要用途是核酸或寡核苷酸 的直接标记， 标记的核酸或寡核苷酸主要有三个用途： 一是作为探针用于核酸杂 交和足迹法分析， 包括 Southern blot, Northern blot, Slot blot, dot blot, Southern- western blot, 原位杂交等； 二是用作核酸体外扩增的引物， 三是用作 DNA序列分 析. 酶蛋白与核酸或寡核苷酸的连接主要是通过化学或物理等方法进行共价交 联， 可通过交联臂将核酸或寡核苷酸的末端基团与酶蛋白的氨基基团或巯基基团 相连（Ruth et ah DNA 1985; 4:93 ) . 检测方法可采用化学、 物理或生物学方法. 根据固相杂交和液相杂交而不同， 前者用 BCIP/NBT为底物的显色法或以 AMPPD 为底物的化学发光法 ( Schaap A， et al. Clin. Chem. 1989， 35:1863-1864 )为好； 后 者以对硝基酚（pNPP ) 为底物为好， 可进行定量测定.
附图说明：
图 1: 耐热碱性磷酸酯酶基因的核苷酸序列及推导所得的氨基酸序列. 氨基 酸序列标在 DNA序列下方（氨基酸残基以三个字母表示） ， 氨基酸序列的 N端用 横线标出了有 26个氨基酸残基的信号肽.
图 2: 克隆有 FD-TAP基因的高表达质粒 pTAP503示意图.
图 3: FD-TAP的耐热性. 测定方法为酶溶液在 95'C水浴保温不同时间， 再在 测定酶活力. 图 4: FD-TAP的最适温度.
图 5: pH对 FD-TAP活力的影响.
通过以下实例对本发明予以详细说明， 但决不是以此限制本发明。
在实例中所用的缩写符号：
TE: 1 Ommol L Tris-HCl,lmmol/L EDTA, pH8.0
TH: 0.3%蛋白胨， 0.3%酵母抽提物， 0.2% NaCl，pH7.0
LB: 1%蛋白胨， 0.5°/。酵母抽提物， 1% NaCl, pH7.0
2χΥΤ: 1.6%蛋白胨， 1%酵母抽提物， 0.5%NaCl， ρΗ7.0
FD-TAP: 来源于 77ie 7m« sp.FD3041的耐热碱性磷酸酯酶. 实例 1:
Figure imgf000009_0001
sp.FD3041分离染色体 DNA
在 200ml TH液体培养基中 70'C培养 ¾er/m« sp.FD3041(可购自复华实业有限 公司， 上海， 中国)。 菌液经离心收集菌体， 用 12ml TE缓冲液悬浮， 加人 lml含 10mg/ml溶菌酶的 TE缓冲液， 于 37'C水浴 2小时， 加人 1.5ml含 10°/。 十二垸基肌酸 钠（Sarcosyl )、 lmg/ml蛋白酶 K的 TE缓冲液， 37'C水浴 1小时， 用酚、 氯仿 /异戌 醇（24: 1 )各抽提两次， 水相加人 1/10体积的 3mol/L 乙酸钠， 用 2倍体积的乙醇 沉淀 DNA, 用玻棒绕出絮状沉淀物， 真空干燥后溶解于 3ml TE缓冲液中， 加入 50 μ1 10ιη§/πι1 RNase A, 用氯仿 /异戌醇抽提一次， 乙醇沉淀后溶于 TE缓冲液. 实例 2 克隆编码耐热碱性磷酸酯酶的 DNA片段
用 Sau3AI部分消化 2(^g的 7¾mn«i sp.FD3041染色体 DNA, 用低熔点琼脂糖 胶电泳回收 3 ~ 10kb的 DNA片段. 为防止自连， 其粘性末端用 Klenow大片段和 dGTP、 dATP进行两个碱基的部分填补； 载体 pUCl 18用 Sail完全酶切， 回收大片 段， 同样用 Klenow大片段和 dCTP、 dTTP部分填补粘性末端的两个碱基， 填补后 的染色体和载体 DNA的粘性末端可以互连. 经 T4连接酶连接后转化 .co// TGl，在 含 ITPG、 X-gal和氨苄青霉素（ lOO g/ml ) 的 LB平板上挑选白色的重组转化子. 共获得 1.2万个转化子， 经抽提质粒鉴定， 约 85%含有 3 - 10kb的插入片段. 由此构 建了 rhermus sp.FD3041的染色体基因文库.
采用菌落原位碱性磷酸酯酶显色法对上述基因文库进行筛选， 方法如下： 将 菌落转移到 3MM滤纸上， 浸于破菌缓冲液 (lmol/L二乙醇胺， 1%SDS)， 于 85Ό水 浴保温 10min，再浸于反应缓冲液 (6mmol/L pNPP， lmol/L二乙醇胺， 1°/。SDS)中置 70 :水浴保温 10min， 菌落颜色转黄者为阳性. 通过筛选， 获得 5个阳性克隆. 对 其中的 1个克隆 (PTAP362)进行物理图谱和测定部分缺失的质粒的 TAP活性， 将 FD-TAP基因定位于 2kb的 DNA片段上.
用 Sanger双脱氧链终止法测定 DNA序列， 获得的核苷酸序列为 2030bp (图 1 )。 经计算机分析， FD-TAP的基因长度为 1506bp， （3+ %为68.2%, 密码子第三 位碱基 0+ %为92.7%, 符合栖热菌属的基因特征. 该基因编码一个由 501个氨基 酸残基组成的原始酶蛋白， 在 N端有一个 26个氨基酸残基的信号肽序列， 成热的 酶蛋白分子由 475个氨基酸残基组成. FD-TAP基因的 DNA序列和编码蛋白质的氨 基酸序列见图 1. 实例 3 .FD-TAP基因的亚克隆和高表达：
分别在 FD-TAP基因的起始密码子处及终止密码子处设计了引物， 引物的 5' 端分别带有 Ndel和 BamHI酶切位点，通过 PCR技术从质粒 pTAP118B中扩增获得了 成熟的 FD-TAP的基因序列. 经过酶切将基因克隆到高表达载体 pJLA503, 经转化 phoA基因缺陷菌株 Mph44后，在含氨苄青霉素的 LB平板上， 用菌落原位显 色法筛选重组的转化子，结果 50%的转化子为表达 FD-TAP的阳性克隆， 对其中一 个克隆所含的重组质粒（记为 pTAP503，见图 2 )进行 DNA序列分析， 表明基因内 未发生突变.
E. coli Mph44 (pTAP503F)经 30'C液体培养后于 42'C诱导 10小时， SDS- PAGE表明在约 53kDa处有酶的表达产物. 约占总蛋白的 10 % . 实例 4. 重组 FD-TAP的分离纯化：
挑一环 Mph44(pTAP503)菌株，接种于 2 χ ΥΤ培养基（含氨苄青霉素 100ug/ml)，30'C振荡培养过夜作为种子液，取种子液按 2%接种量转接于 2 χ ΥΤ培养 基， 30'C振荡培养至菌体 Α600=0.4 ~ 0.6时，再在 42'C中培养 10小时， 离心收集菌 体，用缓冲液 A(50mmol/L Tris pH8.8, 甘油， 10mmol/L P -巯基乙醇)中， 冰浴中超 声破碎 (总处理时间为 400s，脉冲作用时间 Is,脉冲间隔时间 Is,输出功率 25%)破菌 液于 15，000rpm离心 15分钟，弃沉淀，收集上清. 在上清液中慢慢加入 PEI以去 除核酸 (PEI浓度为 0.04%), 15，000rpm离心 15分钟， 弃沉淀， 收集上清. 上清中加 入 NaCl至 NaCl终浓度为 0.8mmol/L，然后在 70 'C水浴中热变性处理 30分 钟， l，5000rpm离心 15分钟,去除细胞总蛋白中不耐热的杂蛋白，收集上清.在热变性 后的上清中逐步加入固体硫酸铵，达 60°/。的饱和度， 4 'C搅拌 1小时， 12,000rpm离心 20min,弃上清，沉淀用 1/8体积的缓冲液 B(10mmol/L Na2HP04— NaH2P04， pH6.8，5%甘油， lOmmol/L P -巯基乙醇)溶解， 4'C对相同缓冲液透析除盐。透析后的 蛋白样品用 CM Sepharose Flast Flow柱进行离子交换层析，上样后用缓冲液 B洗脱 至样品 A280恢复至基线，再用 NaCl梯度 (0― 0.5mol/L)进行线性梯度洗脱，分管收集 有酶活峰，每管 1.5ml，SDS-I>AGE电泳分析蛋白质的纯度，合并纯蛋白峰， 冷冻抽干 后 -20'C保存。 实例 5. FD-TAP酶活性测定和性质：
取待测酶液 ΙΟμΙ加人 ΙΟΟΟμΙ反应体系中 （反应体系： 6mmol/L, 对硝基酚， lmol/L二乙醇胺， pHl l.6 )，70'C水浴 10分钟， 加 990μΕ 5%三氯醋酸液终止反应， 在 UV260上测定 OD405处生成物的吸光值。 酶活单位的定义： 在 70'C， pHl 1.6时， 每分钟催化产生 lpmol/L的对硝基酚的酶量定为一个酶活单位. 酶单位 =A405x 2/(18.8x10). 其中 2为反应总体积， 10为反应时间， 对硝基酚在 405nm处的克分子 消光系数为 18.8 06.
FD-TAP的部分酶学性质：
最适反应温度： 70'C (图 3 )
耐热性： 在 50 mmol/L Tris, pH8.8， 25'C的系统中， 95 'C中水浴保温 30分钟， 仍保留原来酶活的 90%以上. （图 4 )
最适 pH: pH12 (图 5 ) 实例 6. FD-TAP基因的部分缺失：
分别于 FD-TAP基因的不同位置设计引物， 扩增大小不同的片段： 79—1506、 79→1416、 79→960、 271→480、 271 330。 上游引物的 5'端带有 Ndel 酶切位点， 下游引物带有终止密码子和 Bamffl酶切位点， 用 PCR从质粒 pTAP118B 中扩增所需的 DNA片段. 经过酶切将这些 DNA片段克隆于高表达载体 pJLA503， 经转化 E oli Mph44和筛选重组的转化子， 获得含有上述各种 DNA片段的阳性克 隆. 诱导表达和分离纯化重组的多肽后， 对其酶学性质进行了研究， 表明这些多 肽仍具有与完整的 FD-TAP相似的性质和特征. 序列表
(1)一般信息：
①申请人： 复旦大学
(ii)发明名称:一种耐热碱性磷酸酯酶及其表达
(iii)序列数目： 2
(V)本申请资料：
(A) 申请号：
(vi) 在先申请资料：
(A) 申请号： CN97106724.4
(B) 申请日： 13-11月 -1997
(2)SEQ ID NO. 1的信息
(i)序列特征
(A)长度： 1506碱基
(B)类型： 核酸
(C)链性： 单链
(D)拓扑结构： 线性
(ii)分子类型： 寡核苷酸
(xi)序列描述： SEQ ID NO ： 1
1 ATG AAG CGA AGG GAC ATC CTG AAA GGT GGC CTG GCT GCG GGG GCC
46 CTG GCC CTC CTG CCC CGG GGC CAT ACC CAG GGG GCT CTG CAG AAC
91 CAG CCT TCC TTG GGA AGG CGG TAC CGC AAC CTC ATC GTC TTC GTC
136 TAC GAC GGG TTT TCC TGG GAG GAC TAC GCC ATC GCC CAG GCC TAC
181 GCC CGG AGG CGG CAG GGC CGG GTT CTC GCC CTG GAG CGC CTC CTC
226 GCC CGC TAC CCC AAC GGG CTC ATC AAC ACC TAC AGC CTC ACC AGC
271 TAC GTC ACC GAG TCC AGC GCC GCG GGG AAC GCC TTC TCC TGC GGG
316 GTG AAG ACG GTG AAC GGG GGG CTC GCC ATC CAC GCC GAC GGG ACC
361 CCC CTC AAG CCC 丌 C TTC GCC GCG GCC AAG GAG GCG GGG AAG GCC
406 GTG GGG CTC GTG ACC ACC ACC ACC GTC ACC CAC GCC ACC CCG GCG
451 AGC 丌 C GTG GTG TCC AAT CCC GAC CGG AAC GCC GAG GAG AGG ATC 496 GCC GAG CAG TAC CTG GAG TTC GGG GCC GAG GTG TAC CTT GGG GGC
541 GGG GAC CGC πτ TTC AAC CCC GCC AGG CGC AAG GAC GGG AAG GAC
586 CTC TAC GCC GCC TTC GCC GCC AAG GGG TAC GGG GTG GTG CGC ACC
631 CCC GAG GAG CTC GCC CGT TCC AAC GCC ACC CGG CTC CTG GGC GTC
676 TTC GCC GAC GGC CAC GTG CCC TAC GAG A丌 GAC CGC CGC 丌 C CAG
721 GGC CTT GGG GTG CCG AGC CTC AAG GAA ATG GTC CAG GCC GCT丌 G
766 CCC CGG CTT GCC GCC CAC CGC GGG GGC TTC GTC CTT CAG GTG GAA
811 GCG GGG CGG ATT GAC CAC GCC AAC CAT TTG AAC GAC GCC GGG GCC
856 ACC CTT TGG GAC GTG CTG GCG GCG GAC GAG GTC TTG GAG CTT CTC
901 ACC GCC 丌 C GTG GAC CGG AAC CCG GAC ACC CTC CTC CTC GTG GTC
946 TCG GAC CAC GCC ACC GGG GTG GGG GCC CTC TAC GGG GCG GGC CGG
991 AGC TAC CTG GAG AGC TCC GTG GGC ATT GAC CTC CTG GGG GCG CAA
1036 AAG GCC AGC TTT GAG TAC ATG CGC CGC GTC TTG GGC TCG GCC CCC
1081 GAT GCT GCC CAG GTG AAG GAG GCC TAC CAG ACC CTG AAG GGG GTC
1126 TCC CTC ACG GAC GAG GAG GCG CAG ATG GTG GTC CGG GCC ATC CGC
1171 GAG CGG GTC TAC TGG CCT GAT GCC GTG CGC CAG GGC ATC CAG CCC
1216 GAA AAC ACC ATG GCC TGG GCC ATG GTG CAG AAG AAC GCC AGC AAG
1261 CCC GAC CGG CCC AAC ATC GGC TGG AGC TCT GGG CAG CAC ACG GCG
1306 AGC CCC GTC ATC CTC CTC CTC TAC GGC CAG GGC CTG CGC 丌 C GTC
1351 CAG C丌 GGC CTG GTG GAC AAC ACC CAC GTG 丌 C CGC CTG ATG GGC
1396 GAG GCC CTG AAC CTC CGC TAC CAG AAC CCG GTG ATG AGC GAG GAG
1441 GAG GCC CTG GAG ATC CTC AAG GCC AGG CCC CAG GGG ATG CGC CAC
1486 CCC GAG GAC GTC TGG GCC TAA
(2)SEQ ID N0. 2的信息：
(i)序列特征：
(A)长度： 501个氨基酸
(B)类型： 氨基酸
(D)拓扑结构： 线性
(ii)分子类型： 多肽
(xi)序列描述： SEQ ID N0. 2 :
1 Met Lys Arg Arg Asp H e Leu Lys Gly Gly Leu Al a Al a Gly Al a 16 Leu Al a Leu Leu Pro Arg Gly Hi s Thr Gi n Gly Al a Leu Gi n Asn Gin Pro Ser Leu Gly Arg Arg Tyr Arg Asn Leu He Val Phe Val
T r Asp Gly Phe Ser Trp Glu Asp Tyr Ala lie Ala Gin Ala Tyr
Ala Arg Arg Arg Gin Gly Arg Val Leu Ala Leu Glu Arg Leu Leu
Ala Arg Tyr Pro Asn Gly Leu lie Asn Thr Tyr Ser Leu Thr Ser
Tyr Val Thr Glu Ser Ser Ala Ala Gly Asn Ala Phe Ser Cys Gly
Val Lys Thr Val Asn Gly Gly Leu Ala lie His Ala Asp Gly Thr
Pro Leu Lys Pro Phe Phe Ala Ala Ala Lys Glu Ala Gly Lys Ala
Val Gly Leu Val Thr Thr Thr Thr Val Thr His Ala Thr Pro Ala
Ser Phe Val Val Ser Asn Pro Asp Arg Asn Ala Glu Glu Arg lie
Ala Glu Gin Tyr Leu Glu Phe Gly Ala Glu Val T r Leu Gly Gly
Gly Asp Arg Phe Phe Asn Pro Ala Arg Arg Lys Asp Gly Lys Asp
Leu Ty「 Ala Ala Phe Ala Ala Lys Gly Tyr Gly Val Val Arg Thr
Pro Glu Glu Leu Ala Arg Ser Asn Ala Thr Arg Leu Leu Gly Val
Phe Ala Asp Gly His Val Pro Tyr Glu lie Asp Arg Arg Phe Gin
Gly Leu Gly Val Pro Ser Leu Lys Glu Met Val Gin Ala Ala Leu
Pro Arg Leu Ala Ala His Arg Gly Gly Phe Val Leu Gin Val Glu
Ala Gly Arg He Asp His Ala Asn His Leu Asn Asp Ala Gly Ala
Thr Leu Trp Asp Val Leu Ala Ala Asp Glu Val Leu Glu Leu Leu
Thr Ala Phe Val Asp Arg Asn Pro Asp Thr Leu Leu Leu Val Val
Ser Asp His Ala Thr Gly Val Gly Ala Leu T r Gly Ala Gly Arg
Ser Tyr Leu Glu Ser Ser Val Gly lie Asp Leu Leu Gly Ala Gin
Lys Ala Ser Phe Glu Tyr Met Arg Arg Val Leu Gly Ser Ala Pro
Asp Ala Ala Gin Val Lys Glu Ala Tyr Gin Thr Leu Lys Gly Val
Ser Leu Thr Asp Glu Glu Ala Gin Met Val Val Arg Ala He Arg
Glu Arg Val Tyr Trp Pro Asp Ala Val Arg Gin Gly lie Gin Pro
Glu Asn Thr Met Ala Trp Ala Met Val Gin Lys Asn Ala Ser Lys
Pro Asp Arg Pro Asn lie Gly Trp Ser Ser Gly Gin His Thr Ala
Ser Pro Val He Leu Leu Leu Tyr Gly Gin Gly Leu Arg Phe Val
Gin Leu Gly Leu Val Asp Asn Thr His Val Phe Arg Leu Met Gly
Glu Ala Leu Asn Leu Arg T r Gin Asn Pro Val Met Ser Glu Glu
Glu Ala Leu Glu He Leu Lys Ala Arg Pro Gin Gly Met Arg His
Pro Glu Asp Val Trp Ala

Claims

权 利 要 求 书 1一种耐热碱性磷酸酯酶， 其特征在于它的氨基酸序列与下列氨基酸序列同 源或基本同源。 1 Met L.ys Arg Arg Asp He Leu Lys Gly Gly Leu Ala Ala Gly Ala 16 Leu Ala Leu Leu Pro Arg Gly His Thr Gin Gly Ala Leu Gin Asn31 Gin Pro Ser Leu Gly Arg Arg Tyr Arg Asn Leu He Val Phe Val46 Tyr Asp Gly Phe Ser Trp Glu Asp Tyr Ala He Ala Gin Ala Tyr61 Ala Arg Arg Arg Gin Gly Arg Val Leu Ala Leu Glu Arg Leu Leu76 Ala Arg Tyr Pro Asn Gly Leu lie Asn Thr Tyr Ser Leu Thr Ser91 Tyr Val Thr Glu Ser Ser Ala Ala Gly Asn Ala Phe Ser Cys Gly106 Val Lys Thr Val Asn Gly Gly Leu Ala He His Ala Asp Gly Thr121 Pro Leu Lys Pro Phe Phe Ala Ala Ala Lys Glu Ala Gly Lys Ala136 Val Gly Leu Val Thr Thr Thr Thr Val Thr His Ala Thr Pro Ala151 Ser Phe Val Val Ser Asn Pro Asp Arg Asn Ala Glu Glu Arg He166 Ala Glu Gin Tyr Leu Glu Phe Gly Ala Glu Val Tyr Leu Gly Gly181 Gly Asp Arg Phe Phe Asn Pro Ala Arg Arg Lys Asp Gly Lys Asp196 Leu Tyr Ala Ala Phe Ala Ala Lys Gly Tyr Gly Val Val Arg Thr211 Pro Glu Glu Leu Ala Arg Ser Asn Ala Thr Arg Leu Leu Gly Val226 Phe Ala Asp Gly His Val Pro Tyr Glu He Asp Arg Arg Phe Gin241 Gly Leu Gly Val Pro Ser Leu Lys Glu Met Val Gin Ala Ala Leu256 Pro Arg Leu Ala Ala His Arg Gly Gly Phe Val Leu Gin Val Glu271 Ala Gly Arg lie Asp His Ala Asn His Leu Asn Asp Ala Gly Ala286 Thr Leu Trp Asp Val Leu Ala Ala Asp Glu Val Leu Glu Leu Leu301 Thr Ala Phe Val Asp Arg Asn Pro Asp Thr Leu Leu Leu Val Val316 Ser Asp His Ala Thr Gly Val Gly Ala Leu Tyr Gly Ala Gly Arg331 Ser Tyr Leu Glu Ser Ser Val Gly He Asp Leu Leu Gly Ala Gin346 Lys Ala Ser Phe Glu Tyr Met Arg Arg Val Leu Gly Ser Ala Pro361 Asp Ala Ala Gin Val Lys Glu Ala T r Gin Thr Leu Lys Gly Val376 Ser Leu Thr Asp Glu Glu Ala Gin Met Val Val Arg Ala lie Arg391 Glu Arg Val T r Trp Pro Asp Ala Val Arg Gin Gly lie Gin Pro406 Glu Asn Thr Met Ala Trp Ala Met Val Gin Lys Asn Ala Ser Lys 421 Pro Asp Arg Pro Asn li e Gly Trp Ser Ser Gly Gi n Hi s Thr Al a436 Ser Pro Val H e Leu Leu Leu T r Gly Gi n Gly Leu Arg Phe Val451 Gi n Leu Gly Leu Val Asp Asn Thr Hi s Val Phe Arg Leu Met Gly466 Gl u Al a Leu Asn Leu Arg T r Gi n Asn Pro Val Met Ser Gl u Gl u481 Gl u Al a Leu Gl u li e Leu Lys Al a Arg Pro Gi n Gly Met Arg His496 Pro Gl u Asp Val Trp Al a 2.根据权利要求 1所述的耐热碱性磷酸酯酶， 其特征在于：
(1) 由与下述核苷酸序列同源或基本同源的 DNA片段所编码:
1 ATG AAG CGA AGG GAC ATC CTG AAA GGT GGC CTG GCT GCG GGG GCC 6 CTG GCC CTC CTG CCC CGG GGC CAT ACC CAG GGG GCT CTG CAG AAC 1 CAG CCT TCC TTG GGA AGG CGG TAC CGC AAC CTC ATC GTC 丌 C GTC 6 TAC GAC GGG TTT TCC TGG GAG GAC TAC GCC ATC GCC CAG GCC TAC 1 GCC CGG AGG CGG CAG GGC CGG 6TT CTC GCC CTG GAG CGC CTC CTC 6 GCC CGC TAC CCC AAC GGG CTC ATC AAC ACC TAC AGC CTC ACC AGC 1 TAC GTC ACC GAG TCC AGC GCC GCG GGG AAC GCC TTC TCC TGC GGG 6 GTG AAG ACG GTG AAC GGG GGG CTC GCC ATC CAC GCC GAC GGG ACC 1 CCC CTC AAG CCC TTC TTC GCC GCG GCC AAG GAG GCG GGG AAG GCC 6 GTG GGG CTC GTG ACC ACC ACC ACC GTC ACC CAC GCC ACC CCG GCG 1 AGC TTC GTG GTG TCC AAT CCC GAC CGG AAC GCC GAG GAG AGG ATC 6 GCC GAG CAG TAC CTG GAG TTC GGG GCC GAG GTG TAC CTT GGG GGC 1 GGG GAC CGC TTT 丌 C AAC CCC GCC AGG CGC AAG GAC GGG AAG GAC 6 CTC TAC GCC GCC 丌 C GCC GCC AAG GGG TAC GGG GTG GTG CGC ACC 1 CCC GAG GAG CTC GCC CGT TCC AAC GCC ACC CGG CTC CTG GGC GTC 6 丌 C GCC GAC GGC CAC GTG CCC TAC GAG ATT GAC CGC CGC TTC CAG 1 GGC CTT GGG GTG CCG AGC CTC AAG GAA ATG GTC CAG GCC GCT 丌 G 6 CCC CGG CTT GCC GCC CAC CGC GGG GGC TTC GTC CTT CAG GTG GAA 1 GCG GGG CGG ATT GAC CAC GCC AAC CAT TTG AAC GAC GCC GGG GCC 6 ACC C丌 TGG GAC GTG CTG GCG GCG GAC GAG GTC TTG GAG C丌 CTC 1 ACC GCC 丌 C GTG GAC CGG AAC CCG GAC ACC CTC CTC CTC GTG GTC 6 TCG GAC CAC GCC ACC GGG GTG GGG GCC CTC TAC GGG GCG GGC CGG 1 AGC TAC CTG GAG AGC TCC GTG GGC ATT GAC CTC CTG GGG GCG CAA 1036 AAG GCC AGC 丌 T GAG TAC ATG CGC CGC GTC TTG GGC TCG GCC CCC
1081 GAT GCT GCC CAG GTG AAG GAG GCC TAC CAG ACC CTG AAG GGG GTC
1126 TCC CTC ACG GAC GAG GAG GCG CAG ATG GTG GTC CGG GCC ATC CGC
1171 GAG CGG GTC TAC TGG CCT GAT GCC GTG CGC CAG GGC ATC CAG CCC
1216 GAA AAC ACC ATG GCC TGG GCC ATG GTG CAG AAG AAC GCC AGC AAG
1261 CCC GAC CGG CCC AAC ATC GGC TGG AGC TCT GGG CAG CAC ACG GCG
1306 AGC CCC GTC ATC CTC CTC CTC TAC GGC CAG GGC CTG CGC TTC GTC
1351 CAG CTT GGC CTG GTG GAC AAC ACC CAC GTG TTC CGC CTG ATG GGC
1396 GAG GCC CTG AAC CTC CGC TAC CAG AAC CCG GTG ATG AGC GAG GAG
1441 GAG GCC CTG GAG ATC CTC AAG GCC AGG CCC CAG GGG ATG CGC CAC
1486 CCC GAG GAC GTC TGG GCC TAA
(2) 由上述 DNA片段经部分缺失后的 DNA片段所编码。
3. 一种能够在宿主中表达耐热碱性磷酸酯酶的重组载体， 其特征在于该载体 含有权利要求 2所述 DNA片段的一个或多个拷贝 .
4.根据权利要求 3所述的重组载体， 其特征在于该载体为原核载体。
5.根据权利要求 4所述的重组载体， 其特征在于该载体为质粒。
6.根据权利要求 3-5中任一项所述的重组载体， 其中插入的 DNA片段在一启动 子的控制之下.
7.—种获得权利要求 2所述的 DNA片段的方法， 其特征在于其步骤如下：
( 1 )分离原核生物的染色体 DNA, 并用合适的限制性内切酶进行消化，
( 2 )将消化后的 DNA整合到一载体中， 用该重组载体转化合适的宿主， 并以 此方式构建染色体基因文库；
( 3 )用通常的方法筛选步骤（2 ) 的基因文库；
( 4 )对步骤（3 )筛选的阳性克隆进行分析.
8.—种生产如权利要求 1所述的耐热碱性磷酸酯酶的方法， 其特征在于其步骤 如下：
( 1 ) 用权利要求 2所述的 DNA片段或权利要求 3 - 6中任一项所述的重组载体 转化合适的宿主；
( 2 )在合适的培养基中培养转化后的宿主；
( 3 )从培养基或细胞中分离纯化蛋白质.
PCT/CN1998/000272 1997-11-13 1998-11-13 Phosphoesterase alcaline calorifuge et son expression WO1999027115A1 (fr)

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US09/530,851 US6649390B1 (en) 1997-11-13 1998-11-13 Thermophilic alkaline phosphoesterase and its expression
AU11402/99A AU1140299A (en) 1997-11-13 1998-11-13 A thermophilic alkaline phosphoesterase and its expression
EP98954109A EP1038964A4 (en) 1997-11-13 1998-11-13 THERMOPHILES, ALKALINE PHOSPHOESTERASE AND THEIR EXPRESSION

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CN97106724.4A CN1178835A (zh) 1997-11-13 1997-11-13 一种耐热碱性磷酸酯酶及其表达
CN97106724.4 1997-11-13

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CN1178835A (zh) 1998-04-15
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AU1140299A (en) 1999-06-15
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