CN1060212C - 盘状网柱菌二肽氨肽酶,其分离方法及用途 - Google Patents
盘状网柱菌二肽氨肽酶,其分离方法及用途 Download PDFInfo
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- CN1060212C CN1060212C CN93118452A CN93118452A CN1060212C CN 1060212 C CN1060212 C CN 1060212C CN 93118452 A CN93118452 A CN 93118452A CN 93118452 A CN93118452 A CN 93118452A CN 1060212 C CN1060212 C CN 1060212C
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- ddap
- dipeptide
- met
- dap
- precursor polypeptide
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Abstract
本发明涉及从细胞粘液霉菌-盘状网柱菌中分离得到一种新的二肽氨肽酶。这个新的DAP酶，即dDAP，具有类似DAP-Ⅰ与DAP-Ⅱ两者的活性，但在物理及其它酶学特征上与这些酶有很明显的区别。本发明还涉及利用dDAP酶从重组产生的前体蛋白及肽类的N-末端移去二肽的方法。此外，本发明还涉及从盘状网柱菌中分离和纯化dDAP酶的方法。
Description
本发明涉及生物技术领域。更确切地说，本发明涉及从粘液霉菌盘状网柱菌中分离得到的一种二肽氨肽酶，它可用于加工重组产生的生物化合物。
盘状网柱菌是一种原始的真核微生物，通常被称为粘液霉菌，或者更确切地称为细胞粘液霉菌。这个名称是从肉眼观察到的该微生物的两个极端状态而来的。当活跃生长时，盘状网柱菌生长为单细胞变形虫。在这个阶段它们没有细胞壁，因此它们外表为一层薄膜(或者粘液)。在固体培养基并处于饥饿状态下，各个独立的细胞聚集形成一个集落。这个集落呈现一种多细胞生物体的特征，因为它以蛞蝓的方式移动，然后进行分化。它后面的细胞形成一个足，前面的细胞形成一个柄，而中间的细胞形成一个果实体。这个生物体天然是在泥土和粪肥的表面发现的。野生型变形虫只通过摄取(吞噬)整个的细菌来获得营养，因此它们有时被称为是食肉的。盘状网柱菌的无外来污染的突变体己被分离出来，它们能在无“食物”细菌的共培养下生长，所以能生长在可溶培养基中。本发明涉及从盘状网柱菌中分离的二肽氨肽酶。
二肽氨肽酶(DAP)是水解氨基端倒数第二个肽键的酶，从肽的蛋白质的未封闭的氨基端释放二肽。目前有四类二肽氨肽酶(称做DAP-Ⅰ、DAP-Ⅱ、DAP-Ⅲ和DAP-Ⅳ)，它们的区别在于其物理性质及其催化裂解不同的多肽氨基端序列的速率的差异。DAP-Ⅰ是一种从蛋白质和肽类未封闭的氨基末端催化释放出许多二肽复合物的相对非特异性的DAP。如果出现的二肽是X-Pro、Arg-X或者Lys-X(X为任何一种氨基酸)，那么DAP-Ⅰ几乎不显示活性。DAP-Ⅱ对氨基端二肽顺序为Arg-X或Lys-X的优先，其次为X-Pro，而对其余大多数二肽复合物DAP-Ⅱ的裂解速率显著降低。DAP-Ⅲ似乎对氨基端二肽顺序为Arg-Arg和Lys-Lys的具有倾向性。DAP-Ⅳ对二肽顺序为X-Pro的显示最高的水解活性。DAP酶，尤其是DAP-Ⅰ与DAP-Ⅳ在处理加工蛋白质方面已显示出其用途。本发明涉及盘状网柱菌中的一种新DAP，它用于加工以偶数个氨基酸N-末端延伸的重组蛋白。
本发明涉及从细胞粘液霉菌-盘状网柱菌中分离的一种新二肽氨肽酶。这个新的二肽氨肽酶-dDAP显示出有些类似DAP-Ⅰ和DAP-Ⅲ两者的活性，但在物理和其它的酶特征方面又与它们有显著区别。本发明还涉及利用dDAP酶从重组产生的前体蛋白或前体多肽的N-末端移去二肽的方法。本发明的dDAP酶可用于从多肽的N-末端移去单个二肽，也可用于从前体多肽的N-末端顺序移去一个以上的二肽。此外，本发明还涉及从盘状网柱菌的培养物中分离纯化此dDAP酶的方法。
就本发明目的而言，在本文说明书和权利要求书所用的下列术语及缩写定义如下：
dDAP为一种从盘状网柱菌中分离得到的二肽氨肽酶，用GFpNA作底物证明其最适pH为pH3．5，用超离心分析测得其分子量约为22 5，000道尔顿，通过SDS-聚丙烯酰胺凝胶电泳得知其单个亚基的分子量约为66，000道尔顿。
GFpNA为甘氨酰-苯丙氨酰-对硝基苯胺。
前体多肽为一种重组产生的多肽，它包括由从有意义的期望的多肽的氨基末端延伸的偶数个氨基酸。
加工后多肽为一种多肽，其中N-末端二肽或多个二肽已被移去，生成有意义的目的多肽。
RRBNA为精氨酰-精氨酰-β-萘胺
本公开应用的所有氨基酸缩写均被美国专利与商标局在37C．F．R．§1．822(b)(1990)中认可。
本发明是针对一种从细胞粘液霉菌盘状网柱菌中分离出的一种二肽氨肽酶，dDAP。dDAP酶显示出裂解未封闭氨基末端顺序的倾向，而它们过去习惯上由DAP-Ⅰ和DAP-Ⅲ共同完成，即便如此，dDAP在物理及其它酶学特性上与这些酶存在明显区别。dDAP以GFpNA为底物测得其最适pH约为pH3．5，天然dDAP分子量约为225，000道尔顿，而单个亚基的分子量约为66，000道尔顿。凝集素亲合色谱表明dDAP很可能是一个糖蛋白。dDAP酶能从合成的底物GFpNA和RRBNA以及其它许多合成的和重组产生的多肽上移去二肽。
已知的DAP-Ⅰ酶已从多种动物及动物组织中分离得到。新酶dDAP是从盘状网柱菌的培养物中分离得到的。DAP-Ⅰ酶需要卤化物和还原剂才有活性。还原剂(如碘代乙酸盐，它修饰半胱氨酸巯基)钝化DAP-Ⅰ酶。 DAP-Ⅰ在pH5-6之间有最理想的活性。与此相反，以GFpNA或RRBNA作底物，dDAP酶的最适pH约为3．5，而且在pH3．5条件下dDAP对蛋白质和肽也具显著活性，但它在pH＞6条件下无明显活性。dDAP酶无需添加还原剂并且在半胱氨酸修饰物(如碘代醋酸盐和连四硫酸盐)的存在下仍具完全活性。dDAP与牛DAP-Ⅰ相似，因为它不能裂解N-末端封闭的肽，但是dDAP又与牛DAP-Ⅰ不同，它对N-末端具有氧化的甲硫氨酸底物仍具活性，而DAP-Ⅰ不能裂解在N-末端具有氧化的甲硫氨酸的底物。此外，与牛DAP-Ⅰ不同，dDAP酶能够容易地裂解RRBNA底物。这种裂解包含裂解Arg-Arg二肽基的氨基末端底物的能力，与哺乳和微生物来源的DAP-Ⅲ酶活性很相似。虽然据报道DAP-Ⅲ酶在碱性范围内具有最适pH，而dDAP在酸性范围具有最有效作用。据SDS-PAGE测定，dDAP亚基分子量约为66，000道尔顿，而哺乳动物DAP-Ⅰ的亚基分子量约为22，000道尔顿。
本发明的dDAP酶对于将前体多肽转变为加工后多肽非常有用。例如，若目的多肽是人类生长激素，那么只需表达此激素的前体(在一实例中，Met-Asp-人类生长激素)，然后将此前体在dDAP的作用下释放出二肽Met-Asp和加工后的目的多肽人生长激素。加工后的肽无需是“天然”的野生型多肽，因为通常希望产生类似物或中间体。 加工前体多肽的方法也是本发明的一个部分。可被dDAP加工的其它前体多肽包括：Met-Arg-人生长激素，Met-Tyr-胰岛素原，Met-Arg-胰岛素原，Met-Arg-胰岛素原类似物(B28Lys，B29 Pro)，Met-Arg-胰岛素原类似物(B10 Asp，des B28-30)和Met-Tyr-胰岛素原类似物(B10 Asp，des B28-30)。胰岛素类似物(B28 Lys，B29 Pro)公开在欧洲专利申请序号为90301224．3中，而胰岛素类似物(B10 Asp，des B28-30)公开在欧洲专利申请序号为92305678．2中。此外，dDAP还可用于从前体多肽N-末端顺序地移去一个以上的二肽。用牛DAP-Ⅰ加工Met-Arg-胰岛素原及其类似物公开于美国专利申请5，126，249号中(1992年6月30日授予Becker等人)。所有这些教导均作为参考文献并入本文中。
利用dDAP酶从前体蛋白质移去二肽是有利的，因为dDAP最适pH约为3．5，这就允许反应在许多前体多肽可溶的酸性pH范围内进行。再者，在中性或更高pH条件下一些前体多肽的转化会导致链间二硫聚合物或底物多聚体生成水平增加，而这伴随着产物产量损失。这个现象叫作二硫化物混杂(scrambling)，尤其在使用牛DAP-Ⅰ时很麻烦，因为DAP-Ⅰ在反应混合物中需要还原试剂的存在，如β-巯基乙醇或半胱氨酸。而且在酸性pH范围里甲硫氨的残基的氧化发生率较低。此外，从盘状网柱菌发酵培养物中分离的酶比从动物来源大批量生产的酶更经济，因为发酵技术允许较高浓度的产物存在以及酶的再生。避开动物来源的酶，主要考虑到大量高纯原料的恒定来源。盘状网柱茵Ax3(ATCC 28368)的发酵，然后通过离心、阴离子交换色谱、巯水相互作用色谱、容积排阻色谱可得到高度纯化的dDAP酶溶液，它可以贮存或立即用于加工前体多肽。从发酵液体培养基中分离和纯化dDAP也是本专利的一个部分。
将前体多肽转变为加工后多肽，可在一个较大的温度范围、pH范围和时间阶段内完成。反应一般在使pH保持在约2．5-5．5的缓冲的水性介质中进行。介质的pH范围优选大约3．0-4．5，最好在约3．0-3．5，最适pH可能根据底物不同而有微小变动。例如，加工GFpNA和GRpNA的速率在pH约3．5时最快，而加工Met-Asp-hGH的速率在pH约3．0-3．5时较迅速。加工Arg-Arg-BNA的速率在pH约4．5时最快。熟练的技术人员会认识到任何特定反应的最适pH是由诸如所给的前体多肽和酶的稳定性、溶解度等因素决定的。在某些情况下，可采用如尿素、十二烷基硫酸钠、胍等增溶剂。
加工反应可在从仅几秒钟到几天的时间范围内进行。优选大约1分钟到24小时，最优选大约1小时到8小时。熟练的技术人员会认识到反应时间能容易地根据任意需要的前体多肽或加工后多肽所需的参数来调整。
加工反应的温度还能根据所给底物进行调整。反应一般在大约15℃～45℃进行，优选大约20℃-37℃，最优选约25℃-37℃。再者，熟练的技术人员会容易的认识到反应的pH、温度和时间参数会因需要的前体多肽和加工后多肽的需要而有所变动。
只要能够保持所需要的pH范围，可使用任何范围的缓冲试剂。代表性的缓冲试剂实例是：磷酸钠、乙酸钠、柠檬酸钠、甘氨酸等。缓冲试剂优选为乙酸钠、磷酸钠和甘氨酸。
本发明中使用的前体多肽一般是通过重组DNA技术制备的。在制备中目的前体多肽的核苷酸编码顺序是利用这类合成的常规技术制备的。这些方法一般包括目的编码序列片段和它的互补序列寡核苷酸编码的制备。设计的寡核苷酸以提供编码序列的一个片段与互补序列的两个片段的重叠，反过来也是这样。将寡核苷酸配对连结，最后得到目的基因顺序。
此顺序插入克隆载体，其位置在能表达产物的编码区。一个合适的克隆载体至少包含表达控制顺序的一个部分。
下列实施例用于举例说明此发明。它们决不是对本发明的限制。
实施例1
盘状网柱菌的发酵
盘状网柱菌Ax3的冻干培养液是从注册号为ATCC 28368的马里兰州罗克维尔美国典型培养物收集中心获得的，然后接种到几种浓度的琼脂培养皿上(1．2％Difco Bacto琼脂)，其中包括用缓冲液处理的酵母提取蛋白胨培养基，组成为(克／升)：Difco酵母提取物(7．15)，Difco Bacto胨(14．3)，Na2HPO4(0．51)和KH2PO4(0．49)，并加入单独灭菌的葡萄糖(终浓度为10克／升)，再用NaOH或H2SO4调整至最终pH为6．5(+／-0．1)。相同的培养基(没有琼脂)用于体积小于一升的液体培养生长。琼脂培养皿在21℃～24℃培养3-5天。从培养基上收获孢子团，小心不要将与Ax3培养基一起冻干的“食物细菌”也取出来，然后将孢子团移入3ml缓冲液处理的酵母提取蛋白胨液体培养基中，并在21℃～24℃轻微振荡培养。此后，盘状网柱菌细胞通过连续地转移到体积逐渐增大的用缓冲液处理的酵母提取蛋白胨液体培养基中增殖。连续的每步转移都是在细胞浓度超过2×106／ml时并稀释大约10-25倍。液体培养基总是在轻度摇动下在21℃～24℃培养。
搅动的发酵一般在一种相似的培养基中进行，即用浓度为2-14．3克／升的大豆蛋白胨(如植物蛋白胨或Marcor大豆蛋白胨)来代替起初的酵母提取蛋白胨培养基。通常从配有1到3个转速为40-150转的Rushon涡轮推进器、工作体积为10到2000升的发酵器中收获。温度控制在22±1℃，空气流动控制在每体积液体培养基0．1到0．5体积空气，排气压力保持在3-5p．s．i．。有些发酵pH用硫酸控制在6．4，有些通过变化的搅动和空气流动将溶解氧控制在40-60％。在细胞的处理和发酵中小心减少切变，因为它们在生长过程中是没有细胞壁的变形虫。
通常，盘状网柱菌Ax3的搅动培养液在12-36小时内加倍增长。溶解氧逐渐减少(当不控制时)并在细胞密度停止增长后某一时间开始回升。最终细胞密度在3×106／ml到5×107／ml范围内，伴随着氧的转移明显限制在较低的最大细胞密度的发酵过程中。
样品不定时地取出并分析其细胞密度和GFpNA的活性(参见下文实施例3)。当高于约5×105／ml时，用Petroff-Hauser计数板来测定细胞密度。通常，在发酵过程中GFpNA水解活性逐渐增加。达到最大细胞密度后的2到4天可观察到最大的dDAP活性。所有的液体培养基在4℃贮存或-20℃冻存，然后解冻并分析活性。发酵通过冷却到10℃以下并用连续流动离心机去掉细胞获得产品。
实施例2
dDAP的制备
A．细胞的去除和浓缩
从盘状网柱菌发酵液体培养基中对dDAP的最初纯化包括细胞去除和浓缩步骤。细胞的去除是在Western States离心机上用连续流动离心完成的。无细胞培养基通过用能去除50，000分子量以下物质的膜进行切向流动超滤，被浓缩约20倍。保留物从超滤室中排出，用50mM三羟甲基氨基甲烷(Tris)缓冲液(pH7)冲洗超滤室以回收残留的dDAP。保留物与洗下的样品合并形成最终浓缩物。在进一步处理前，合并液可在-20℃冷冻保存几个月。
B．澄清
冷冻的最终浓缩物在室温解冻约12小时。一旦解冻，在第一次柱色谱步骤前，最终浓缩物要进行澄清。澄清操作由离心和随后的5微米滤膜过滤完成。将澄清的最终浓缩物调节pH至7．0，在等待离子交换色谱时可于4～10℃下保存不超过12小时。
C．离子交换色谱法
dDAP纯化过程的首次柱色谱法是阴离子交换色谱，用Pharmacia Q-琼脂糖快流速树脂(FFQ)。交换柱用50mM tris缓冲液(pH=7)平衡。澄清的无细胞浓缩物按每升树脂对应60升未浓缩的发酵培养液以线性流速为50cm／hr的速率上样。结果每升树脂可获得60克蛋白(蛋白量以牛血清白蛋白作标准，通过Pierce BCA蛋白分析测定)。每升FFQ树脂上样的dDAP活性约为250单位。无细胞浓缩物电导率约为5mMHOS／cm。上样完毕后，FFQ树脂用三倍柱体积的平衡缓冲液冲洗。用10倍柱体积的0-1M NaCl，50mMTris(Ph7)以流速为50cm／hr对树脂进行线性梯度洗脱得到dDAP活性物，每组分为0．1柱体积。FFQ柱进一步用3倍柱体积的pH7．050mM Tris(内含1．0M NaCl)进一步洗脱。流出物通过电导率和在280nm处的吸收进行监测。各组分通过在pH3．5时其裂解GFpNA比色底物的能力来分析dDAP活性。将包含大约90％洗脱下的总dDAP活性物的各组分合并组成主流池。dDAP活性物以大约两倍柱体积的单峰被洗脱下来。主流池用10％(V／V)的HCl酸化到pH3．5。FFQ经酸化的主流池在4℃最多贮存两天。
D．疏水相互作用色谱法
FFQ酸化的主流池再用疏水相互作用色谱法(HIC)纯化。用药用苯基琼脂糖凝胶(Pharmacia Phenyl Sepharose)快流速树脂，柱体积为阴离子交换柱的三分之一。每升树脂上样约650单位活性物。每升树脂的蛋白上样量是4克(在280nm，一个吸收单位等于1mg／ml蛋白)。每升加入140克硫酸铵制备FFQ主流液上到HIC柱。所上样调节到pH3．5，最终电导率约为90mMHOS／cm。HIC柱用PH3．5，50mM柠檬酸盐平衡，其中每升至少含有140g硫酸铵。以40cm／hr的线流速上料，该树脂至少用三倍柱体积的平衡缓冲液冲洗。dDAP活性物用10倍于柱体积的含140g／L到0g／L硫酸铵、pH3．5的50mM柠檬酸盐溶液以40cm／hr的速率进行线性梯度洗脱。柱子进一步用至少三倍柱体积的50mM柠檬酸盐(pH3．5)溶液洗脱。每组分为0．1倍柱体积，流出液用电导率和280nm处的吸收来监测。流分的dDAP活性通过它们在pH3．5时裂解GFpNA的能力来测定。将包含大约90％洗脱下的总dDAP活性物各组分合并组成主流池。洗脱的为单峰的dDAP活性物约为柱体积的2倍。主流池用10％(V／V)HCl或10％(W／V)NaOH调节至pH3．5。在后面的处理过程前，HIC主流池在4℃保存不超过一天。
E．体积排阻色谱法
HIC主流池进一步用S-200琼脂糖凝胶体积排阻色谱法(SEC)处理。所用柱的体积为HIC柱的两倍，柱床高78cm。通过可去除10，000道尔顿分子量的膜在超滤单元中浓缩HIC主流物，制备用于SEC柱的HIC主流物。HIC主流物浓缩到SEC柱体积的2．5％，并把超滤室内的保留物排出，用等于2．5％SEC柱体积、pH3．5的50mM柠檬酸盐缓冲液冲洗超滤室。保留物与冲洗液合并，形成最终浓缩物，用10％(V／V)HCl或10％(w／V）NaOH调节pH至3．5。最终浓缩物的电导率约为30mM HOS／cm。SEC柱用50mM乙酸、20mM氯化钠(pH3．5)平衡，其电导率约为2mMHOS／cm。最终浓缩物以15cm／hr的线性流速上到SEC柱中，dDAP活性物用一倍柱体积的平衡缓冲液洗脱下来，每组分是柱体积的0．02倍。洗脱物用电导率和在280nm处的吸收来监测，通过其在pH3．5时裂解GFpNA的能力来测定组分中dDAP活性。将包含大约90％洗脱的总dDAP活性物和组分合并组成主流池。dDAP活性物以单峰形式洗脱下来，约为柱体积的0．08倍。SEC主流池可以在4℃保存几个月。
用阴离子交换色谱法、疏水相互作用色谱法及体积排阻色谱法的组合来进行dDAP纯化，得到的物质在SDS-PAGE上以一条主带迁移。此带在凝胶上迁移的位置相当于标准牛血清白蛋白分子量(66 KD)。蛋白用ISS普鲁兰(Pro-blue)染色。迁移图谱不受样品制备过程中含不含0．1M DTT(100℃加热五分钟)的影响。DAP-Ⅰ(来源于牛)亚基分子量用SDS-PAGE测得约为22，000道尔顿。
实施例3
dDAP的活性
A．转化反应
1．GF-PNA的裂解
DAP的活性通常是通过跟踪生色底物Gly-Phe-对硝基苯胺(GF-PNA)的裂解来监测的。典型的检测是在1．0ml调节到pH3．5的4mM GFpNA溶液中把酶稀释11倍进行的。GF二肽裂解速率在37℃通过测定405nm处吸收值的增加来检测。
在这些条件下，每单位活性引起每分钟0．90oD值变化。假定405nm处游离PNA的消光系数为9．9mM-1cm-1，可以估计出单位／ml值。
以GFpNA做底物，用碘乙酰胺、连四硫酸钾以及已知的抑制DAP-Ⅰ活性的巯基修饰试剂作抑制剂，将它们对dDAP的抑制与对牛DAP-Ⅰ的抑制相对照。dDAP或牛脾DAF-Ⅰ的样品在100mM三羟甲基氨基甲烷缓冲液中配成pH7的抑制剂终浓度为0，0．5，5．0或50mM体系中，室温孵育15分钟。孵育液然后用pH3．5的4mM GFpNA稀释21倍。通过测定37℃时在405nm处吸收值的增加来监测裂解速率。当反应体系含5mM碘代乙酰胺时，牛DAP-Ⅰ裂解GFpNA的速率降低大于90％，而含5mM连四硫酸钾抑制95％。没有迹象表明，dDAP明显受实验中所用任何浓度的碘代乙酰胺和连四硫酸钾抑制。
dDAP裂解GFpNA的最适pH是在由0．5三羟甲基氨基甲烷、磷酸盐和柠檬酸盐组成的缓冲液中，用10％HCl或10％NaOH将pH调节至3-8的几个不同pH缓冲液中测得的。dDAP酶在含100mM半胱胺和10mM 10mM NaCl的缓冲液中稀释20倍，牛DAP-Ⅰ在同样缓冲液中稀释200倍。GFpNA底物(4mM)溶于2％DMF中。在一微滴定平板上，将0．025ml各种pH值的三羟甲基氨基甲烷／磷酸盐／柠檬酸盐缓冲液与0．1ml稀释的酶和0．1ml底物溶液混合，30分钟内，在平板阅读器上于410nm处测定吸收值增加的速率。结果表明，dDAP裂解DFpNA的最适pH在3．5到4．0之间。
2．Gly-Arg-pNA的裂解
在pH为5的50mM乙酸、50mM甘氨酸缓冲液中配制4mMGly-Arg-pNA(GR-pNA)。用HCl或NaOH将pH调至5．1-2．3几个不同的pH值。向180ml上述pH的底物缓冲液中加入5mldDAP(终浓度为49毫单位／ml)，在410nm处的吸收值增加速率用平板阅读器监测，并与反应液的pH相对照。与GF-PNA底物相似，GR-PNA底物在大约3．5有一最适pH值在pH低于2．5或高于5时，此酶对该底物几乎没有活性。
3．Arg-Arg-β-萘酰胺(RR-BNA)的裂解
将约0．25mM RR-BNA或0．25mM苄氧羰基-RR-BNA(Z-RR-BNA)溶于pH3．5的100mM乙酸中或溶于pH5．0的100mM柠檬酸盐缓冲液中。向2ml底物中加入dDAP或牛DAP-Ⅰ(约每毫升溶液含15毫单位)，通过监测340nm激发后410nm处的荧光增加来检测裂解的速率。牛DAP-Ⅰ不能裂解任何一种底物，而dDAP出人意料地能有效裂解RR-BNA底物。dDAP不能裂解封闭氨基基团的Z-RR-BNA底物，这更证明了dDAP是一个DAP酶。采用含有50mM乙酸和50mM柠檬酸盐的缓冲体系，通过监测RR-BNA的裂解速率，探测出最适合RR-BNA裂解的pH值。使用HCl或NaOH得到各种pH，每种pH缓冲液取1．5ml与0．5ml1mM的RR-BNA贮存液混合得2．0ml(RR-BNA终浓度约0．25mM)，加入dDAP(约15mM／ml)，测定其裂解速率，在探测的整个范围(pH3．5-5．7)，观察到的裂解RR-BNA的最适pH约为4．5。这时该酶有显著的活性。这一惊人结果提示dDAP具有DAP-Ⅲ的某些性质。
熟练的技术人员会认识到，底物裂解的最适pH不仅依赖于酶，而且也与底物自身有关。也就是说，与被切下的二肽和指示基团自身的组成有关。例如，使用dDAP，Gly-Arg-pNA的最适pH大约是3．5，而Gly-Arg-7-氨基-4-甲基香豆素(GR-AMC)裂解的最适pH约为5，提示报道的基团会影响裂解的性质。
B．合成的八肽及十肽的转化
八肽Met-Asp-Phc-Pro-Ala-Met-Ser-Leu用pH3．5的50mM乙酸溶解，其浓度为4mM。此溶液用dDAP(10mM／ml)按1∶1稀释，并在室温孵育6小时，用含1％磷酸的7M尿素稀释20倍，中止反应。中止后的样品用高压反相色谱法(HPLC)来分析。裂解产物与八肽、Met-Asp二肽及Phe-Pro-Ala-Met-Ser-Leu六肽标准品比较。dDAP容易地从未封闭的八肽的氨基端切去Met-Asp二肽，但不易切下暴露出来的Phe-Pro二肽。
合成的十肽Met-Arg-Met-Tyr-Phe-Val-Asn-Gln-His-Leu用pH3．5的100mM甘氨酸配制成1．7mM的贮备液。向1．5ml贮备液中加入8ml 6．4mM／ml的dDAP(用pH3．5的100mM甘氨酸配制)，每小时取5ml该反应液直接注入反相HPLC色谱系统检测裂解产物，将Met-Arg和met-Tyr二肽以及Met-Tyr-Phe-Val-Asn-Gln-His-Leu和Phe-Val-Asn-Gln-His-Leu肽分别单独注入做为对照。dDAP容易地从十肽上切下Met-Arg二肽,同样容易地切下暴露出来的Met-Tyr二肽。这表明二肽能够顺次从氨基端被dDAP切下。
C．Met-Asp-人生长激素的转化
Met-Asp-人生长激素(Met-Asp-hGH)在大肠杆菌的胞浆中以不溶性蛋白生产出来。该不溶性蛋白被溶解，然后对折生成具有配对的二硫化物的Met-Asp-hGH，并用离子交换色谱法进行纯化。这一制备过程中溶剂是可变换的，并被调节到pH3．5。将Met-Asp-hGH温热到37℃，测定其在280nm处的吸收。以每毫克Met-Asp-hGH 6毫单位的量加入dDAP。转化反应在37℃进行，搅拌大约4-6个小时。采用较少的酶、较低的温度或较低的Met-Asp-hGH浓度，反应进程可被放慢而不会有损害。加入更多的酶、提高Met-Asp-hGH的浓度或升高反应温度可使反应速率增加。转化反应的进行由反相色谱来监测。搅拌下，快速加入NaOH至pH8，并加入30％(V／V)乙腈，使转化反应得以中止。dDAP处理后得到的人生长激素反应产物要经受一组广泛的分析步骤，包括肽图谱、N末端序列分析、质谱、氨基酸分析以及反相色谱(HPLC)。所有数据表明，由dDAP得到了可靠的人生长激素。
D．Met-Arg-人胰岛素原的转化
Met-Arg-人胰岛素原(Met-Arg-hPI)作为一种不溶蛋白在大肠杆菌胞浆中产生。此不溶蛋白溶解于7M尿素溶液中，由离子交换色谱法纯化。Met-Art-hPI被硫解(sulfitolyzed)、溶剂交换并对折生成天然的二硫键配对物和天然的三级结构，并进一步用反相色谱纯化。用过氧化氢从met-Arg-hPI得到氧化的甲硫氨酰基(Met(o)-Arg-hPI，随后用反相色谱纯化并冷冻干燥。
得到的Met-Arg-hPI为约24mg／ml(在pH3．5约20mM甘氨酸缓冲液中)，大约2．4mg此原料用0．1％毫单位的dDAP在pH3．5下孵育，反应可在室温进行。定期地取出等份试样并用10％磷酸稀释。将稀释液注入中性反相HPLC系统，检测Met-Arg-hPI或Met(o)-Arg-hPI的消失以及随后的hPI产物。另外，等份试样被稀释成合适的稀释液，以便通过HPLC检测二肽Met-Arg或Met(o)-Arg。大约60％的Met-Arg-hPI在8小时后转变成hPI。在对于Met(o)-Arg-hPI的转化实验中出乎意料地看到了相似的结果，即产生了hPI，两个底物的裂解速率相似。这一结果是令人吃惊的，因为牛DAP-Ⅰ似乎不能裂解hPI的Met(o)-X-衍生物，其中X为Arg、Phe和Tyr。反相色谱分析结果也表明以二肽met-Arg作对照，Met-Art二肽从Met-Arg-hPI中释放出来。用met(o)-Arg-hPI作底物，在Met-Arg二肽区出现的一个峰，它可能是二肽Met(o)-Arg。dDAP裂解氧化的Met(o)-X底物的能力比不能完成这一裂解的酶具有显著的加工优点。
E．Met-Arg-人胰岛素原类似物的转化
dDAP酶也可用来有效地转化折叠的Met-Arg-胰岛素原类似物(B28Lys，B29Pro)及Met-Arg-胰岛素原类似物(B10Asp，desB28-B30)。这些反应的进行同前述对Met-Arg-hPI转化的解释基本一致。
Claims (9)
1．从盘状网柱菌中分离得到的二肽氨肽酶，其天然分子量约为225，000道尔顿，亚基分子量约为66，000道尔顿，以Gly-Phe-对硝基苯胺为底物，其最适pH约为pH3．5。
2．从前体多肽中移去氨基端二肽的方法，所说的方法包括在适宜于二肽氨肽酶作用的条件下，将前体多肽与权利要求1的二肽氨肽酶接触，以逐步移去前体多肽的氨基端二肽。
3．权利要求2的方法，其中前体多肽选自人胰岛素原前体、人生长激素前体和人胰岛素原类似物前体。
4．权利要求3的方法，其中所述的前体多肽与所述的二肽氨肽酶接触大约1分钟到24小时。
5．权利要求4的方法，其中将所述的前体多肽在约为pH2．5到pH5．5的溶液中与二肽氨肽酶接触。
6．权利要求5的方法，其中将所述的前体多肽在pH约为3．5的溶液中与二肽氨肽酶接触。
7．权利要求3的方法，其中将所述的前体多肽在约15℃到45℃之间与二肽氨肽酶接触。
8．权利要求2的方法，其中所述的二肽的N-末端氨基酸是氧化的甲硫氨酸。
9．从盘状网柱菌培养物中分离权利要求1的二肽氨肽酶的方法，所述的方法包括：
a)从培养液体中获得无细胞培养液；
b)将所得的培养液经过阴离子交换色谱；
c)将b)步的洗脱液进行疏水相互作用色谱；
d)将c)步洗脱液进行体积排阻色谱。
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US95553992A | 1992-10-01 | 1992-10-01 | |
| US955,539 | 1992-10-01 |
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| Publication Number | Publication Date |
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| CN1085253A CN1085253A (zh) | 1994-04-13 |
| CN1060212C true CN1060212C (zh) | 2001-01-03 |
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| CN93118452A Expired - Fee Related CN1060212C (zh) | 1992-10-01 | 1993-09-30 | 盘状网柱菌二肽氨肽酶,其分离方法及用途 |
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| EP (2) | EP1094118A1 (zh) |
| JP (1) | JP3550409B2 (zh) |
| KR (1) | KR100291528B1 (zh) |
| CN (1) | CN1060212C (zh) |
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| AU (1) | AU669735B2 (zh) |
| BR (1) | BR9303949A (zh) |
| CA (1) | CA2107279A1 (zh) |
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| DK (1) | DK0595476T3 (zh) |
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| FI (1) | FI934305L (zh) |
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| NO (1) | NO933503L (zh) |
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| PL (1) | PL176930B1 (zh) |
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| US5573923A (en) * | 1993-12-22 | 1996-11-12 | Eli Lilly And Company | Method for removing N-terminal dipeptides from precursor polypeptides with immobilized dipeptidylaminopeptidase from dictyostelium discoideum |
| US5614379A (en) * | 1995-04-26 | 1997-03-25 | Eli Lilly And Company | Process for preparing anti-obesity protein |
| WO2000017332A1 (en) * | 1998-09-21 | 2000-03-30 | Eli Lilly And Company | Production of soluble recombinant trypsinogen analogs |
| US6794159B1 (en) | 1999-04-30 | 2004-09-21 | Pharmacia Corporation | Method of removing n-terminal alanine residues from polypeptides with aeromonas aminopeptidase |
| US6743600B1 (en) | 1999-04-30 | 2004-06-01 | Monsanto Technologies Llc | Method of removing N-terminal alanine residues from polypeptides with Aeromonas aminopeptidase |
| US8129139B2 (en) | 2009-07-13 | 2012-03-06 | Allergan, Inc. | Process for obtaining botulinum neurotoxin |
| US20110237509A1 (en) * | 2010-02-17 | 2011-09-29 | Elona Biotechnologies | hGH AND METHODS FOR PREPARING hGH |
| EP3947667A4 (en) * | 2019-03-26 | 2022-12-28 | Encodia, Inc. | MODIFIED CLEAVAGES, USES AND CORRESPONDING KITS |
| US11427814B2 (en) | 2019-03-26 | 2022-08-30 | Encodia, Inc. | Modified cleavases, uses thereof and related kits |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| EP0397420A1 (en) * | 1989-05-09 | 1990-11-14 | Eli Lilly And Company | Enzymatic removal of a protein amino-terminal sequence |
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| DK55685A (da) * | 1985-02-07 | 1986-08-08 | Nordisk Gentofte | Enzym eller enzymkompleks med proteolytisk aktivitet |
| DE3519218A1 (de) * | 1985-05-29 | 1986-12-04 | Boehringer Mannheim Gmbh, 6800 Mannheim | H(pfeil abwaerts)2(pfeil abwaerts)o(pfeil abwaerts)2(pfeil abwaerts)-bildende sarcosinoxidase, ihre herstellung und verwendung |
| US4894439A (en) * | 1986-05-22 | 1990-01-16 | Cetus Corporation | N-terminal derivatives of tumor necrosis factor purified by microporous PTFE membranes |
| US5177003A (en) * | 1987-04-16 | 1993-01-05 | Phillips Petroleum Company | Flavored protein products derived from Candida utilis |
| US4929700A (en) * | 1987-04-16 | 1990-05-29 | Cetus Corporation | Production of purified, biologically active, bacterially produced recombinant human CSF-1 |
| US5075222A (en) * | 1988-05-27 | 1991-12-24 | Synergen, Inc. | Interleukin-1 inhibitors |
| JPH023698A (ja) * | 1988-06-20 | 1990-01-09 | Denki Kagaku Kogyo Kk | ヒトリンホトキシンに対するモノクローナル抗体及びそれら抗体を産生するハイブリドーマ、並びにそれら抗体を用いたヒトリンホトキシンの精製方法、測定方法及び測定試薬 |
| US5208218A (en) * | 1988-09-19 | 1993-05-04 | Ludwig Institute For Cancer Research | T cell growth factor glycoproteins |
| US5175251A (en) * | 1989-05-04 | 1992-12-29 | Sri International | Antimetastatic peptides with laminin activity |
| US5179196A (en) * | 1989-05-04 | 1993-01-12 | Sri International | Purification of proteins employing ctap-iii fusions |
| US5173409A (en) * | 1989-12-08 | 1992-12-22 | Ecogen Inc. | Recovery of bt endotoxin protein from lysed cell mixtures |
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| EP0397420A1 (en) * | 1989-05-09 | 1990-11-14 | Eli Lilly And Company | Enzymatic removal of a protein amino-terminal sequence |
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