CN108018323A - 一种腺苷酸基琥珀酸或盐的制备方法 - Google Patents
一种腺苷酸基琥珀酸或盐的制备方法 Download PDFInfo
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- CN108018323A CN108018323A CN201810030628.4A CN201810030628A CN108018323A CN 108018323 A CN108018323 A CN 108018323A CN 201810030628 A CN201810030628 A CN 201810030628A CN 108018323 A CN108018323 A CN 108018323A
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- OFBHPPMPBOJXRT-UHFFFAOYSA-N adenylosuccinic acid Natural products OC1C(O)C(COP(O)(O)=O)OC1N1C2=NC=NC(NC(CC(O)=O)C(O)=O)=C2N=C1 OFBHPPMPBOJXRT-UHFFFAOYSA-N 0.000 title claims abstract description 17
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/38—Nucleosides
- C12P19/40—Nucleosides having a condensed ring system containing a six-membered ring having two nitrogen atoms in the same ring, e.g. purine nucleosides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/16—Purine radicals
- C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
-
- 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
- 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
-
- 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
- C12N9/93—Ligases (6)
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Abstract
本发明属治疗糖脂代谢紊乱疾病候选药物的规模化生产技术领域,为解决现有S‑AMP难以生产,其价格昂贵,难以保证其成药性研究的原料来源问题,提供一种腺苷酸基琥珀酸或盐的制备方法,利用pET‑28‑a载体表达N末端带有His‑tag序列的PhAdSS,以此为催化剂催化肌苷酸IMP、L‑天冬氨酸和三磷酸鸟苷GTP,进行反应合成S‑AMP,反应结束后,用硅胶薄层层析分析产物中的成分,进行硅胶柱层析分离纯化S‑AMP。合成品内s‑AMP含量为61%,回收率约20%。制备流程简单,成本低廉,为建立工业化的S‑AMP合成工艺奠定基础。有望突破国外对S‑AMP的垄断性销售,可大幅度降低S‑AMP成药性研究成本。
Description
技术领域
本发明属于治疗糖脂代谢紊乱疾病候选药物的规模化生产的技术领域,具体涉及一种腺苷酸基琥珀酸或盐的制备方法及其应用。利用深海古细菌来源的酶来催化合成一种天然腺苷类化合物:腺苷酸基琥珀酸(盐),建立了实验室小试制备方法。这种化合物具有促进胰岛素分泌的功能,可作为改善糖脂代谢紊乱疾病的候选药物开展成药性研究。
背景技术
腺苷酸基琥珀酸(盐)[Adenylosuccinic acid (Adenylosuccinate),简称S-AMP]是一种促进胰岛素分泌,使Ⅱ型糖尿病患者胰岛细胞恢复正常的功能的天然腺苷类化合物[1]。前期研究中我们发现S-AMP是AMPK的一个激动剂,它的降脂活性高于降糖活性[2]。因此S-AMP可作为改善糖脂代谢紊乱新药的候选品来开展成药性研究。迄今为止,仅有Sigma公司销售S-AMP (纯度96%,价格约100元/mg,现停产)。目前急需研发一种降低药物研发成本且纯度增大的合成方法,降低S-AMP作为改善糖脂代谢紊乱新药的侯选品来开展成药性研究的成本。
生物体内的腺苷酸基琥珀酸(盐) S-AMP合成反应是在腺苷酸基琥珀酸合成酶(AdSS)的催化下,利用肌苷酸(IMP)和L-天冬氨酸和三磷酸鸟苷(GTP)为原料合成S-AMP和二磷酸鸟苷酸(GDP)。由于IMP、L-天冬氨酸和GTP都是价格低廉的生物化工产品,发现活性高,稳定性好,因此寻找到易制备的AdSS就可开展S-AMP的大量合成。前期研究中发现古细菌Pyrococcus horikoshii OT3体内的AdSS (PhAdSS)符合这些要求[2,3],然而什么样的合成条件能够高效合成S-AMP且得到纯度符合成药性研究要求的分离方法是建立生产工艺的要解决的关键科学问题。
发明内容
本发明为了解决现有腺苷酸基琥珀酸即S-AMP难以生产,其价格昂贵,难以保证其成药性研究的原料来源问题,提供了一种腺苷酸基琥珀酸或盐的制备方法,是利用酶促反应的S-AMP的小试合成及纯化技术,利用PhAdSS建立了S-AMP的小试合成体系,为建立S-AMP的工业化生产工艺奠定了基础。
本发明由如下技术方案实现的:一种腺苷酸基琥珀酸或盐的制备方法,利用大肠杆菌表达体系pET-28-a载体表达N末端带有MGSSHHHHHHSSGLVPRGSH即His-tag序列的PhAdSS,以此重组蛋白为催化剂催化肌苷酸IMP、L-天冬氨酸和三磷酸鸟苷GTP合成S-AMP和GDP,合成反应结束后,利用硅胶薄层层析分析产物中的成分,根据硅胶薄层层析结果进行硅胶柱层析分离纯化S-AMP,然后采用重结晶法进一步提高产物S-AMP的纯度。
具体制备方法为:
(1)催化剂的合成:a、构建表达质粒pET-28-a-PhAdSS:在浓度分别为1µg/ml 的pET-28-a和pET-19-b-PhAdSS溶液中分别加入限制性内切酶NdeI和BamHI,37℃下反应2 小时后用1%的琼脂糖凝胶电泳进行产物分离,100 V电压下电泳40分钟后,从琼脂糖凝胶中回收pET-28-a和PhAdSS;将pET-28-a和回收的 PhAdSS的cDNA混合,加入T4DNA连接酶反应溶液,37℃下反应16 小时后,将反应生成的pET-28-a-PhAdSS质粒转化到大肠杆菌DH5α菌种内,37℃恒温箱中在含有50µg/ml 硫酸卡那霉素的LB平板培养基上培养大肠杆菌12 小时活化转化子,用含有50µg/ml 硫酸卡那霉素的LB平板培养基37℃下培养转化子12小时,然后用质粒提取试剂盒提取质粒pET-28-a-PhAdSS;b、His-tagged-PhAdSS的表达和纯化:获得的质粒pET-28-a-PhAdSS转化到大肠杆菌表达菌种BL21ED3内,接种于自动诱导培养基上,37℃培养24小时,PhAdSS在大肠杆菌细胞内表达,培养结束后室温下离心回收大肠杆菌细胞,然后用上样缓冲液悬浮、离心去除沉淀,上清液用Ni-NTA蛋白质层析柱洗脱,回收纯化His-tagged-PhAdSS;
(2)S-AMP合成反应:反应体系体积:0.5 - 5 L;反应体系配方:20 mM GTP;10 mM IMP;11 mL L-天冬氨酸;100µg/L His-tagged-PhAdSS;MgCl2 4 mM,用10 M的NaOH调节反应体系pH为7.5;反应温度70℃,反应时间≥6小时,用硅胶薄层层析方法检测IMP转化率;
(3)合成反应体系成分鉴定:合成反应结束后,用硅胶薄层层析分析产物中的成分,展开剂的配方v/v:异丙醇30%,正丁醇30%,浓度为6.25%的氨水40%;
(4)S-AMP纯化:a、洗脱剂配方的确定:按照硅胶薄层层析法检测让S-AMP和GDP在硅胶层上迁移速率最大的展开剂配方;b、样品准备:合成反应完成后调节反应溶液pH值至2.9,室温下放置至少10小时,让过量的L-天冬氨酸析出;过滤去除固体后,上清液转移至蒸发皿内,在水浴锅上用蒸气浴法加热蒸发皿让样品体积浓缩至原来体积的10%,溶液中有不溶物生成过滤去除,或者将溶液完全蒸干,用不超过硅胶柱层析所进样体积的去离子水充分溶解析出的固形物,不溶物过滤去除;c、硅胶层析柱的制备:用10 M NaOH水溶液调节样品溶液的pH至10,用分离样品总质量50倍的200-300目H硅胶进行层析,用无水乙醇湿法装柱得到硅胶层析柱,硅胶柱内硅胶填充高度低于20cm;d、上样:用分离样品总质量5倍的60-100目的上样硅胶吸附分离样品,然后将吸附样品的上样硅胶加入到层析柱内,高度低于0.5cm;e、洗脱:加入硅胶柱体积的1.5倍的流动相溶液,流动相溶液成分为v/v:乙醇60%、NH3浓度为6.25%的氨水40%; f、回收样品:室温下按照每瓶50ml回收流出液,回收的流出液用硅胶薄层层析法检测,展开极为v/v:异丙醇30%,正丁醇30%,NH3浓度为6.25%的氨水40%;g、回收纯化的S-AMP:合并S-AMP为单一成分的流出液,用10 M NaOH水溶液调节pH值为10,旋转蒸发仪去除有机溶剂和氨,剩余水溶液用37%盐酸调节pH值至3,空气浴上蒸干,超纯水溶解获得的固体,过滤去除溶剂,空气浴或室温干燥;f、重结晶S-AMP:用体积比为60%乙醇作为溶剂,在37℃恒温箱内配成饱和溶液,降低温度至室温,然后置于-20℃冰箱内,静置10小时以上,S-AMP重结晶,在室温下抽滤去除液体,结晶室温干燥即得纯度为95%的成品。
步骤(1)中所述PhAdSS在大肠杆菌细胞内表达培养结束后,培养液在离心力4000g离心20min回收大肠杆菌细胞;每升培养基内回收的大肠杆菌细胞用15 ml的上样缓冲液悬浮,70℃恒温20分种后自然冷却至室温;4℃,12000g心20min去除沉淀;上清液流经柱体积10 ml的Ni-NTA蛋白质层析柱后,用50 ml的上样缓冲液流过Ni-NTA蛋白质层析柱洗去杂质,然后用25 ml的洗脱液让His-tagged-PhAdSS从层析柱上洗脱出来,回收样品用SDS-PAGE检测纯度,用G250染色法测定蛋白质浓度;蛋白质层析在室温下实施;柱层析法纯化His-tagged-PhAdSS的流速为 5 mL/min;所述上样缓冲液为:20 mM、pH 8.0的Tris-HCl,500 mM NaCl, 20 mM咪唑;所述洗脱液为:20 mM、pH 8.0的Tris-HCl,500 mM NaCl, 200mM咪唑。
步骤(4)中硅胶柱内加入流动相前在硅胶层顶部放置1层滤纸。所述自动诱导培养基配方为:每升培养基中含有:胰蛋白胨20 g,酵母提取物10g,六水琥珀酸钠5.4g,二水柠檬酸钠1.5g,甘油15g,葡萄糖0.5g,乳糖2g,十二水和磷酸氢二钠3.55g,磷酸二氢钾3.40g,氯化铵2.68g,硫酸钠0.71g,七水硫酸镁0.50g,六水氯化铁0.03g。其中蛋白胨和酵母提取物购自OXoid公司,乳糖和葡萄糖购自上海国药集团,其他试剂均由北京化工厂生产。
本发明利用紫外分光光度法测定的合成品内s-AMP含量为61%,回收率约20%。质谱图中无IMP的峰出现,证明IMP无残留,样品中的杂质可能来源于反应原料。本发明采用带有N端His-tag的PhAdSS依然可以催化S-AMP的大规模合成。这样的重组蛋白制备流程简单,成本低廉,是一个有工业化应用前景的生物催化剂。用His-tagged-PhAdSS催化合成S-AMP的设备简单,安全性好。纯化S-AMP的硅胶柱层析法简单易行,回收率较好。因此本发明建立了一个低成本的S-AMP小试制备技术,为建立工业化的S-AMP生化合成工艺奠定了基础。本发明推广后有望突破国外对S-AMP的垄断性销售,可以大幅度降低S-AMP成药性研究的成本。
附图说明
图1为S-AMP小试合成装置图;图2为用于催化合成S-AMP的His-tagged PhAdSSS各纯化步骤中样品的SDS-PAGE结果图,图中:1. 蛋白质表达结束后大肠杆菌于70℃恒温后上清液;2. 蛋白质表达结束后大肠杆菌于70℃恒温后沉淀;3. 上清液中不与Ni-NTA结合的成分;4. Ni-NTA层析柱上洗脱的成分;图3为不同浓度的重组PhAdSS催化合成S-AMP的反应体系在不同时间点的薄层层析结果图,硅胶板下部数字表示反应开始的时间;图4为本发明所合成的S-AMP与Sigma公司购买标准品的紫外吸收光谱图;图5为LTQ-Orbitrap质谱仪测定合成样品的质谱图,S-AMP的分子量为462.0655;图6为化合物数据库收录的质谱法测定的S-AMP分子量为462.066 (https://metlin.scripps.edu/metabo_info.php olid=3551);图7为 Sigma公司生产S-AMP的质谱图,测定S-AMP的分子量为463.074。
具体实施方式
一种腺苷酸基琥珀酸或盐的制备方法,利用大肠杆菌表达体系pET-28-a载体表达N末端带有MGSSHHHHHHSSGLVPRGSH 即His-tag序列的PhAdSS,以此重组蛋白为催化剂催化肌苷酸IMP、L-天冬氨酸和三磷酸鸟苷GTP合成S-AMP和GDP,合成反应结束后,利用硅胶薄层层析分析产物中的成分,根据硅胶薄层层析结果进行硅胶柱层析分离纯化S-AMP,然后采用重结晶法进一步提高产物S-AMP的纯度。
具体制备方法为:
(1)构建表达质粒:前期研究中我们利用pET-19-b原核表达载体表达了和纯化PhAdSS,依据已报道PhAdSS的表达和纯化条件,每升培养基可获得5 mg纯度大于95%的PhAdSS[3]。为了减小提高PhAdSS的产量,本项发明改用pET-28-a载体表达N末端带有MGSSHHHHHHSSGLVPRGSH (His-tag)序列的PhAdSS,以此重组蛋白为催化剂开展合成S-AMP研究。
在20µl的 pET-28-a (1µg/ml)和pET-19-b-PhAdSS (1µg/ml)溶液中分别加入限制性内切酶NdeI和BamHI各1µl,37℃下反应2小时后用1%的琼脂糖凝胶电泳进行产物分离。100 V电压下电泳40分钟后,从琼脂糖凝胶中回收pET-28-a和PhAdSS。操作依据DNA胶回收试剂盒的条件开展。
取1µl的pET-28-a和3µl的PhAdSS的cDNA混合,加入0.5µl的T4DNA连接酶和0.5µl的T4DNA连接酶反应溶液,37℃下反应16小时后加入50µl,在反应把生成的pET-28-a-PhAdSS质粒转化到大肠杆菌DH5α菌种内。37℃恒温箱中用LB平板培养基(含有50µg/ml 硫酸卡那霉素)培养大肠杆菌12 小时活得转化子后,用5 mL的LB培养基(含有50µg/ml 硫酸卡那霉素)37℃下培养转化子12小时,用质粒提取试剂盒提取pET-28-a-PhAdSS。质粒提取按照Genstar公司查质粒小量提取试剂盒所述操作条件实施。
(2)His-tagged-PhAdSS的表达和纯化:将质粒转化到大肠杆菌表达菌种BL21ED3内,接种于自动诱导培养基,37℃下培养24小时,让PhAdSS在大肠杆菌细胞内表达。培养结束后,室温下将培养液离心力4000 g离心20分钟回收大肠杆菌细胞。每升培养基内回收的大肠杆菌细胞用15 mL的上样缓冲液 [20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 20 mM咪唑]悬浮,70℃恒温20分种后让样品的温度缓慢降至室温。4℃,12000 g离心力下离心20分钟去除沉淀。上清液流经柱体积10 mL的Ni-NTA蛋白质层析柱后,用50 mL的上样缓冲液流过Ni-NTA蛋白质层析柱以洗去杂质,然后用25 mL的洗脱液 [20 mM Tris-HCl (pH8.0), 500 mM NaCl, 200 mM咪唑]让His-tagged-PhAdSS从层析柱上洗脱出来,回收样品用SDS-PAGE检测纯度,用G250染色法测定蛋白质浓度。蛋白质层析在室温下实施。柱层析法纯化His-tagged-PhAdSS的流速为 5 mL/min。
所述自动诱导培养基配方为:每升培养基中含有:胰蛋白胨20 g,酵母提取物10g,六水琥珀酸钠5.4g,二水柠檬酸钠1.5g,甘油15g,葡萄糖0.5g,乳糖2g,十二水和磷酸氢二钠3.55g,磷酸二氢钾3.40g,氯化铵2.68g,硫酸钠0.71g,七水硫酸镁0.50g,六水氯化铁0.03g。其中蛋白胨和酵母提取物购自OXoid公司,乳糖和葡萄糖购自上海国药集团,其他试剂均由北京化工厂生产。
(3)S-AMP合成反应装置和条件:S-AMP合成装置如图1所示。反应体系体积:0.5-5L;反应体系配方:20 mM GTP;10 mM IMP;11 mL L-天冬氨酸;100µg/L His-tagged-PhAdSS;MgCl2 4 mM,用10 M的NaOH调节反应体系pH为7.5。反应温度70℃。反应时间不低于6小时。用硅胶薄层层析方法检测不同酶浓度的IMP转化率。
(4)合成反应体系成分鉴定:合成反应结束后,利用硅胶薄层层析分析产物中的成分,展开剂的配方(v/v):30%异丙醇,30%正丁醇,40%氨水(浓度6.25%)。
(5)S-AMP的纯化流程:洗脱剂配方的确认,根据分离样品的极性差异,用硅胶薄层层析法检测能让S-AMP和GDP在硅胶层上迁移速率最大的展开剂配方。
合成反应完成后调节反应溶液pH值至2.9,室温下放置至少10小时,让过量的L-天冬氨酸析出。过滤去除固体后,上清液转移至蒸发皿内,在水浴锅上用蒸气浴法加热蒸发皿让样品体积浓缩至原来体积的10%,溶液中有不溶物生成过滤去除。也可将溶液完全蒸干,用不超过步骤(3)进样体积的去离子水充分溶解析出的固形物,不溶物过滤去除。
用10 M NaOH水溶液调节样品溶液的pH值至10,用硅胶柱层析分离各组分。硅胶柱的制备方法如下:称取分离样品总质量50倍的H硅胶(200-300目),用无水乙醇调节成糊状,填充至层析柱内,层析柱直径依据柱体积选择,保证硅胶填充高度不超过20 cm,打开层析柱下部活塞,让无水乙醇尽量流出,硅胶层中不得残留气泡。
用分离硅胶质量十分之一的上样硅胶(60-100目)的吸附分离样品,加入在分离硅胶层上,高度不超过0.5 cm。加入体积相当于硅胶柱体积的1.5倍的流动相溶液。流动相成分(v/v): 60%乙醇,40%氨水(NH3浓度为6.25%)。在硅胶层顶部放上1张滤纸,防止加入流动相时液体扰乱硅胶层。
室温下按每瓶50 ml回收流出液。溶液内成分用硅胶薄层层析检测,展开剂的配方(v/v):30%异丙醇,30%正丁醇,40%氨水(浓度6.25%)。
合并S-AMP为单一成份的流出液,用10 M NaOH水溶液调节pH值为10,用旋转蒸发去除有机溶剂和氨后,将剩余水溶液pH值调节为3,在空气浴上蒸干。用超纯水溶解固体,1L反应体积得到的产物用水量为50 mL,过滤去除溶剂,空气浴上或者室温下干燥。获得的产物实施定性和定量分析。
(6)产物定性分析:紫外分光光度法定性分析:配制浓度1µM的S-AMP标准品和合成品溶液浓度,测定紫外吸收光谱。质谱法测定分子量:取1mg合成品溶解于5 mL甲醇中,用DFS质谱分析仪测定样品中各成分的分子量。
(7)纯度分析:用Sigma公司的标准品配制标准溶液,用紫外分光光度法测定266nm的吸光度,绘制工作曲线,准确配制30µM的合成品溶液,测定266 nm的吸光度后,计算合成品内S-AMP的浓度,计算含量。
(8)重结晶:为了进一步提高S-AMP纯度,用60%(v/v)乙醇水溶液将步骤(5)中获得的固体溶液于37℃恒温箱内配成饱和溶液,先将溶液温度降低到室温,然后放入-20℃冰箱内,放置10小时以上,让S-AMP重结晶,而后在室温下迅速抽滤去除液体。获得的结晶在室温下干燥后按照(7)步骤所述条件测定S-AMP的含量。
实验结果
1.PhAdSS的制备:图2显示纯化后蛋白质样品的SDS-PAGE,His-tagged-PhAdSS是其中最主要的成分,杂质蛋白含量可以忽略不计。用G250染色法测定蛋白质浓度计算获得的蛋白总量,通常情况下从1 L自动诱导培养基可得到不少于20 mg的His-tagged-PhAdSS。
2.合成反应结果:图3显示酶浓度为80µg/ml的反应体系中,尽管反应时间超过20小时,IMP不能完全转化为S-AMP,酶浓度为100µg/ml的反应体系中,反应时间超过6小时,反应体系中IMP基本转化为S-AMP,酶浓度为200µg/ml的反应体系中,反应时间超过2小时,IMP可完整地转化为S-AMP。因此可以认为His-tagged-PhAdSS能使IMP完全转化为S-AMP的最低浓度是100µg/ml。
3.产物定性分析结果:迄今为止无S-AMP的紫外吸收光谱被报道,图4为我们首次测定的S-AMP的紫外吸收光谱。S-AMP的最大吸收波长是266nm,合成品和标准品S-AMP的最大吸收波长相同,吸收光谱峰的形状基本相同。
利用TQD质谱仪的高分辨率阴离子法测定的合成品中S-AMP的分子量为426.0655,和化合物数据库收录的阴离子法质谱法测定的S-AMP分子量为462.066,二者相同,该数值+ l即为S-AMP的分子量,Sigma公司提供的S-AMP的分子量也相同。紫外分光光度法和质谱分析结果证明,S-AMP被成功合成。
4.回收率:利用紫外分光光度法测定的合成品内s-AMP含量为61%,此制备方法回收率约20%。质谱图中无IMP的峰出现,证明IMP无残留,样品中的杂质可能来源于反应原料。此样品重结晶后获得的S-AMP纯度为95%,回收率为12%。
参考文献
【1】Gooding J.R., et al., Adenylosuccinate is an insulin secretagoguederived from glucose-induced purine metabolism. Cell Rep. 2015, 13(1): 157–167.
【2】钱火连,小分子量腺苷酸基琥珀酸合成酶的结构与功能及其产物的药理学研究,中国医学科学院/北京协和医学院2014年硕士论文
【3】 Wang X., et al., Overexpression, purification, crystallization andpreliminary crystallographic studies of a hyperthermophilic adenylosuccinatesynthetase from Pyrococcus horikoshii OT3. Acta Cryst. 2011. F67, 1551–1555。
Claims (5)
1.一种腺苷酸基琥珀酸或盐的制备方法,其特征在于:利用大肠杆菌表达体系pET-28-a载体表达N末端带有MGSSHHHHHHSSGLVPRGSH 即His-tag序列的PhAdSS,以此重组蛋白为催化剂催化肌苷酸IMP、L-天冬氨酸和三磷酸鸟苷GTP合成S-AMP和GDP,合成反应结束后,利用硅胶薄层层析分析产物中的成分,根据硅胶薄层层析结果进行硅胶柱层析分离纯化S-AMP,然后采用重结晶法进一步提高产物S-AMP的纯度。
2.根据权利要求1所述的一种腺苷酸基琥珀酸或盐的制备方法,其特征在于:具体制备方法为:
(1)催化剂的合成:a、构建表达质粒pET-28-a-PhAdSS:在浓度分别为1µg/ml 的pET-28-a和pET-19-b-PhAdSS溶液中分别加入限制性内切酶NdeI和BamHI,37℃下反应2 小时后用1%的琼脂糖凝胶电泳进行产物分离,100 V电压下电泳40分钟后,从琼脂糖凝胶中回收pET-28-a和PhAdSS;将pET-28-a和回收的 PhAdSS的cDNA混合,加入T4DNA连接酶反应溶液,37℃下反应16 小时后,将反应生成的pET-28-a-PhAdSS质粒转化到大肠杆菌DH5α菌种内,37℃恒温箱中在含有50µg/ml 硫酸卡那霉素的LB平板培养基上培养大肠杆菌12 小时活化转化子,用含有50µg/ml 硫酸卡那霉素的LB平板培养基37℃下培养转化子12小时,然后用质粒提取试剂盒提取质粒pET-28-a-PhAdSS;b、His-tagged-PhAdSS的表达和纯化:获得的质粒pET-28-a-PhAdSS转化到大肠杆菌表达菌种BL21ED3内,接种于自动诱导培养基上,37℃培养24小时,PhAdSS在大肠杆菌细胞内表达,培养结束后室温下离心回收大肠杆菌细胞,然后用上样缓冲液悬浮、离心去除沉淀,上清液用Ni-NTA蛋白质层析柱洗脱,回收纯化His-tagged-PhAdSS;
(2)S-AMP合成反应:反应体系体积:0.5- 5 L;反应体系配方:20 mM GTP;10 mM IMP;11 mL L-天冬氨酸;100µg/L His-tagged-PhAdSS;MgCl2 4 mM,用10 M的NaOH调节反应体系pH为7.5;反应温度70℃,反应时间≥6小时,用硅胶薄层层析方法检测IMP转化率;
(3)合成反应体系成分鉴定:合成反应结束后,用硅胶薄层层析分析产物中的成分,展开剂的配方v/v:异丙醇30%,正丁醇30%,浓度为6.25%的氨水40%;
(4)S-AMP纯化:a、洗脱剂配方的确定:按照硅胶薄层层析法检测让S-AMP和GDP在硅胶层上迁移速率最大的展开剂配方;b、样品准备:合成反应完成后调节反应溶液pH值至2.9,室温下放置至少10小时,让过量的L-天冬氨酸析出;过滤去除固体后,上清液转移至蒸发皿内,在水浴锅上用蒸气浴法加热蒸发皿让样品体积浓缩至原来体积的10%,溶液中有不溶物生成过滤去除,或者将溶液完全蒸干,用不超过硅胶柱层析所进样体积的去离子水充分溶解析出的固形物,不溶物过滤去除;c、硅胶层析柱的制备:用10 M NaOH水溶液调节样品溶液的pH至10,用分离样品总质量50倍的200-300目H硅胶进行层析,用无水乙醇湿法装柱得到硅胶层析柱,硅胶柱内硅胶填充高度低于20cm;d、上样:用分离样品总质量5倍的60-100目的上样硅胶吸附分离样品,然后将吸附样品的上样硅胶加入到层析柱内,高度低于0.5cm;e、洗脱:加入硅胶柱体积的1.5倍的流动相溶液,流动相溶液成分为v/v:乙醇60%、NH3浓度为6.25%的氨水40%; f、回收样品:室温下按照每瓶50ml回收流出液,回收的流出液用硅胶薄层层析法检测,展开极为v/v:异丙醇30%,正丁醇30%,NH3浓度为6.25%的氨水40%;g、回收纯化的S-AMP:合并S-AMP为单一成分的流出液,用10 M NaOH水溶液调节pH值为10,旋转蒸发仪去除有机溶剂和氨,剩余水溶液pH值调节至3,空气浴上蒸干,超纯水溶解获得的固体,过滤去除溶剂,空气浴或室温干燥;f、重结晶S-AMP:用体积比为60%的乙醇水溶液作为溶剂,在37℃恒温箱内配成饱和溶液,降低温度至室温,然后置于-20℃冰箱内,静置10小时以上,S-AMP重结晶,在室温下抽滤去除液体,结晶室温干燥即得纯度为95%的成品。
3.根据权利要求2所述的一种腺苷酸基琥珀酸或盐的制备方法,其特征在于:步骤(1)中所述PhAdSS在大肠杆菌细胞内表达培养结束后,培养液在离心力4000 g离心20min回收大肠杆菌细胞;每升培养基内回收的大肠杆菌细胞用15 ml的上样缓冲液悬浮,70℃恒温20分种后自然冷却至室温;4℃,12000 g离心20min去除沉淀;上清液流经柱体积10 ml的Ni-NTA蛋白质层析柱后,用50ml的上样缓冲液流过Ni-NTA蛋白质层析柱洗去杂质,然后用25ml的洗脱液让His-tagged-PhAdSS从层析柱上洗脱出来,回收样品用SDS-PAGE检测纯度,用G250染色法测定蛋白质浓度;蛋白质层析在室温下实施;柱层析法纯化His-tagged-PhAdSS的流速为 5 mL/min;所述上样缓冲液为:20 mM、pH 8.0的Tris-HCl,500 mM NaCl, 20 mM咪唑;所述洗脱液为:20 mM、pH 8.0的Tris-HCl,500 mM NaCl, 200 mM咪唑。
4.根据权利要求2所述的一种腺苷酸基琥珀酸或盐的制备方法,其特征在于:步骤(4)中硅胶柱内加入流动相前在硅胶层顶部放置1层滤纸。
5.根据权利要求2所述的一种腺苷酸基琥珀酸或盐的制备方法,其特征在于:所述自动诱导培养基为:每升培养基中含有:胰蛋白胨20 g,酵母提取物10g,六水琥珀酸钠5.4g,二水柠檬酸钠1.5g,甘油15g,葡萄糖0.5g,乳糖2g,十二水和磷酸氢二钠3.55g,磷酸二氢钾3.40g,氯化铵2.68g,硫酸钠0.71g,七水硫酸镁0.50g,六水氯化铁0.03g。
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