CN1246891A - 添加和/或除去了连接基团的多肽-聚合物偶联物 - Google Patents
添加和/或除去了连接基团的多肽-聚合物偶联物 Download PDFInfo
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- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
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Abstract
本发明涉及已添加和/或除去了位于多肽结构表面上用以偶联聚合分子之一个或者多个连接基团的多肽-聚合物偶联物,制备本发明多肽-聚合物偶联物的方法,以及所述偶联物用于降低含有所述偶联物之组合物的免疫原性和变应原性的用途。
Description
发明领域
本发明涉及添加和/或除去了一个或多个连接基团的多肽-聚合物偶联物,所述基团可用于将聚合分子偶联至该多肽3D结构表面上,制备本发明多肽-聚合物偶联物的方法,所述偶联物在降低免疫原性和变应原性方面的用途,以及含有所述偶联物的组合物。
发明背景
医学领域中,熟知多肽(包括酶)可用于循环系统中,以达到特定的生理学效果。此外,在工业应用领域内,如衣物洗涤、纺织品漂白、个人护理、接触性镜片的清洁、食品和饲料制备中,酶被用作功能性成分。药物和工业应用中的重大差异之一在于,后一类型的应用(即工业应用)中,并不打算使多肽(常常是酶)进入体内的循环系统。
某些多肽和酶的稳定性不能令人满意,而且在某些情况下(取决于接触的方式)可能会引起免疫应答,通常是IgG和/或IgE应答。
今天一般都认识到,当多肽(如酶)与聚合分子偶联时,多肽的稳定性会得到提高,免疫应答会减少。人们确信,免疫应答的降低是由于所偶联的聚合分子遮蔽了多肽表面上负责免疫应答、导致抗体形成的表位所致。
用于偶联多肽和聚合分子的技术在本领域是熟知的。
最合适的商业技术之一早在20世纪70年代早期就已描述(如美国专利no.4,179,337)。所说的专利涉及非免疫原性多肽,如与聚乙二醇(PEG)或聚丙二醇偶联的酶和肽激素。其中至少15%的多肽生理活性得到保持。
英国专利no.1,183,257(Crook等)描述了通过三嗪环将酶与多糖偶联的化学。
另外,用于保持酶-聚合物偶联物之酶促活性的技术在本领域也是已知的。
WO 93/15189(Veronese等)涉及一种通过将蛋白水解酶连接到大分子化的抑制剂上来保持经聚乙二醇修饰的蛋白水解酶活性的方法。该偶联物旨在于医疗应用。
已经发现:将聚合分子连接到多肤上通常会通过干扰多肽与其底物相互作用而降低多肽活性。EP 183 503(Beecham Group PLC)公开了上述概念的一种发展,即提供了结合物,所述结合物含有借助于可逆的连接基团而连接到至少一种水溶性聚合物上的药学上有用的蛋白质。
EP471,125(Kanebo)公开了包含通过三嗪环偶联到多糖上以提高耐热性和保存稳定性的亲本蛋白酶(芽孢杆菌蛋白酶,商品名Esperase)的皮肤护理产品。所利用的偶联技术在上述的英国专利no.1,183,257(Crook等)中有描述。
JP 3083908描述了一种皮肤化妆品材料,该材料中含有来自豚鼠肝脏的、用一种或多种水溶性物质(如PEG、淀粉、纤维素等)修饰过的转谷氨酰胺酶。这种修饰是通过激活聚合分子并将其偶联到所说的酶上而进行的。据称该组合物对皮肤比较温和。
然而,不太可能总是能够很容易地将聚合分子偶联到多肽和酶上。此外,仍然需要有免疫原性和/或变应原性进一步降低的多肽-聚合物偶联物。
发明简述
本发明的目的是提供适于工业和药物应用的改良的多肽-聚合物偶联物。
在本发明内容中,术语“改良的多肽-聚合物偶联物”意指在人体和动物体内的免疫应答有所降低、稳定性有所改善的偶联物。免疫应答取决于激发的方式,下面将进一步描述。
本发明人已经发现,通过在亲本多肽表面上添加和/或除去一个或多个连接基团,以使多肽与聚合分子偶联,即有可能使多肽(如酶)的免疫原性和/或变应原性降低。
药用多肽被直接导入循环系统(即血流)中时,其潜在的危害在于可能会引发免疫应答,主要是IgG、IgA和/或IgM抗体形式的应答。与此相对照,工业用多肽,例如在诸如洗涤剂中用作活性成分的酶,并不欲进入循环系统,与工业用多肽有关的潜在危险在于被吸入后会导致主要是IgE抗体形成形式的变应原性应答。
因此,有关工业用多肽,其潜在危险在于通过吸入、气管内和鼻内呈递多肽引发的呼吸性变应原性。
药用多肽的潜在危险主要在于皮内、静脉内或皮下呈递多肽所导致的免疫原性。
应当明白,降低“免疫原性”和降低“呼吸性变应原性”是两个极其不同的问题,即它们的接触途径不同,两种免疫机理也极其不同:
本发明中所用的术语“免疫原性”可以是指临床试验中的变应性接触皮炎,这是对接触和渗入皮肤的化学物质产生的一种细胞介导的迟发型免疫应答。这种细胞介导的反应也称作迟发型接触性过敏反应(根据Gell和Combs的组织损伤中免疫机制的分类,为第IV型反应)。
术语“变应原性”或“呼吸性变应原性”是在吸入诸如多肽后立即发生的过敏反应(根据Gell和Combs,为抗体介导的I型反应)。
根据本发明,可以提供免疫应答有所降低和/或稳定性有所改善的多肽,其基本上保留了剩余活性。
至少在本发明文中,变应性应答和免疫原性应答用一个术语表示,称为“免疫应答”。
在第一方面,本发明涉及一种多肽-聚合物偶联物,其具有:
a)一个或多个额外的聚合分子偶联至多肽上,所述多肽已被修饰,使得与相应亲本多肽上的可用连接基团数相比,该多肽表面上的连接基团数增加了,和/或
b)少一个或多个聚合分子偶联至多肽上,所述多肽已被修饰,使得与相应亲本多肽上的可用连接基团数相比,该多肽的功能性位点处或附近的连接基团数减少了。
术语“亲本多肽”指的是欲通过偶联聚合分子而进行修饰的多肽。亲本多肽可以是天然存在的(或野生型)多肽,或者是通过任何适当方法制得的其变体。例如,亲本多肽可以是通过在天然存在多肽的氨基酸序列中取代、删除或截短一个或多个氨基酸残基或者添加或插入一个或多个氨基酸残基而进行修饰的天然存在多肽的变体。
在本发明上下文中,“合适的连接基团”指的是多肽表面上能够与目的聚合分子偶联的任何氨基酸残基基团。
优选的连接基团是赖氨酸残基的氨基和N-末端氨基。也可以将聚合分子偶联到多肽链上位于表面的氨基酸残基的羧酸基(-COOH)上。羧酸连接基团可以是天冬氨酸或谷氨酸的羧酸基,以及C-末端羧酸基。
“功能性位点”意指对多肽的性能,如催化活性很重要的任何氨基酸残基和/或辅因子,如丝氨酸蛋白酶内的催化性三联体残基-组氨酸、天冬氨酸和丝氨酸,或者例如过氧化物酶(如Arthromyces ramosus过氧化物酶)内的血红素基团和远端及近端组氨酸。
第二方面,本发明涉及制备改良的多肽-聚合物偶联物的方法,包括以下步骤:a)鉴定出位于所述亲本多肽3D结构表面上的氨基酸残基,b)选择出所述待突变的亲本多肽所述3D结构表面上的目标氨基酸残基。c)i)用具有合适连接基团的氨基酸残基取代步骤b)中选定的一个或多个氨基酸残基或者在所选位点插入所述氨基酸残基,和/或
ii)取代或者删除步骤b)中选定的、功能性位点处或者附近的一个或多个氨基酸残基,d)将聚合分子偶联到突变多肽上。
本发明还涉及本发明偶联物的用途,以及降低药物免疫原性和降低工业产品变应原性的本发明方法。
最后,本发明涉及含有本发明偶联物以及工业产品或药物中使用的其它成分的组合物。
附图简述
图1显示用i)对照,ii)未修饰的脂肪酶变体,iii)脂肪酶变体-SPEG免疫接种5次、每周1次后,抗脂肪酶的血清抗体水平。(X:log(血清稀释度);Y:光密度(490/620))。
发明详述
本发明的目的是提供适于工业用和药用的改良的多肽-聚合物偶联物。
尽管药用和工业用多肽可以很不相同,然而,本发明的原理可以根据亲本多肽的具体类型(即酶、激素肽等)稍作改动。
本发明人提供了改良的多肤-聚合物偶联物,所述偶联物与由相应的亲本多肽制得的偶联物相比,其免疫应答有所降低。
本发明人发现,在亲本多肽表面上添加一个或者多个连接基团,会使多肽(如酶)的免疫原性降低和/或变应原性降低。除此之外,本发明人还发现,除去功能性位点处或者附近的连接基团,有可能保持更高百分比的剩余功能活性。
在第一方面,本发明涉及一种改良的多肽-聚合物偶联物,其具有:
a)一个或多个额外的聚合分子偶联至多肽上,所述多肽已被修饰,使得与相应亲本多肽上的可用连接基团数相比,该多肽表面上的连接基团数增加了,和/或
b)少一个或多个聚合分子偶联至多肽上,所述多肽已被修饰,使得与相应亲本多肽上的可用连接基团数相比,该多肽的功能性位点处或附近的连接基团数减少了。
连接基团的添加和/或除去与否取决于特定的亲本多肽。a)添加连接基团
如果亲本多肽表面上的可用连接基团很少,就有可能需要多肽上有更多的连接基团。通过取代或插入一个或多个氨基酸残基而在亲本多肽表面上添加一个或多个连接基团,使得可连接的聚合分子数比相应的亲本多肽要多一些。与其上偶联了更少量聚合分子的相应偶联物相比,含有更多聚合分子连接于其上的偶联物通常具有更弱的免疫应答。
原则上,可以对多肽表面上、优选不位于功能性位点(如酶的活性位点)处或附近的任何可用氨基酸残基进行取代和/或插入,以提供额外的连接基团。
正如下面将会详细描述的那样,对于免疫应答的降低和多肽本身保持的剩余功能活性百分比而言,额外偶联的聚合分子的位置可能比较重要。
与在相应的亲本多肽基础上制得的偶联物的聚合分子数相比,本发明的偶联物通常有1-25个、优选1-10个或更多个额外的聚合分子偶联至多肽的表面。
然而,添加的连接基团的最佳数目(至少部分)取决于欲被偶联聚合分子遮蔽的亲本多肽表面面积(即分子量),当然还取决于亲本多肽上已有的可用连接基团数。b)除去连接基团
在通过与底物或类似物相互作用而行使其功能的酶或者其它多肽的情况下,如果聚合分子偶联至多肤的功能性位点(即酶的活性位点)处或附近,则聚合分子有可能会阻碍多肽与其底物或类似物之间的相互作用。这很有可能会导致活性降低。
若酶有一个或多个聚合分子偶联至其活性位点处或者附近,则预计会使剩余酶促活性显著丧失。因此,根据本发明,可以将偶联物构建成与在目的亲本酶基础上制备的相应偶联物相比具有更高剩余酶促活性百分比的形式。通过取代和/或删除活性位点处或者附近的连接基团、由此改善催化裂隙内底物的可达性而提高底物亲和性,即可达到这点。
与在相应亲本多肽基础上制备的偶联物的聚合分子数相比,本发明的酶-聚合物偶联物在其活性位点处或者附近偶联的聚合分子可少1-25个、优选少1-10个。
正如下面解释的那样,功能性位点处或者附近是指应无聚合分子偶联至距离功能性位点5范围内、优选8范围内、特别是10范围内。
可以将多肽功能性位点处或者附近的连接基团的除去与在多肽表面其它部分添加连接基团有利地组合起来。
这样,连接基团总数可以不发生变化、增加或者减少。然而,连接基团总数的分布已被改良,这可以通过免疫应答的降低和/或保持的剩余活性百分比得到证实。这样也可提高稳定性。连接基团数
一般说来,连接基团数应与多肽分子量和/或表面积平衡。多肽越大,就应有越多的聚合分子偶联至多肽上,以充分遮蔽负责抗体形成的表位。
因此,若亲本多肽分子相对较小(如1-35kDa),则将偶联的聚合分子(功能性位点之外)总数增加到总数4-20个比较有利。
若亲本多肽分子更大一些,例如35-60kDa,则将偶联的聚合分子(功能性位点之外)数增加到7-40等等比较有利。
在下面表1中列出了所述多肽分子量(Mw)与偶联聚合分子数之间认为比较合适的比率。
表1
降低的免疫应答与保持的剩余酶促活性
亲本多肽分子量(Mw)kDa | 与多肽偶联的聚合分子的数目 |
1-35 | 4-20 |
35-60 | 7-40 |
60-80 | 10-50 |
80-100 | 15-70 |
大于100 | 大于20 |
相比于其它类型的多肽来说,尤其是对于酶,由于酶的活性和底物与酶结构中常以裂缝形式存在的活性位点之间的相互作用有关,因此,在降低免疫应答方面与保持显著的剩余酶促活性之间存在冲突。
不受任何理论的限制,可以想见,酶-聚合物偶联物的酶促活性损失可能是由于特别大和/或重的聚合分子以空间阻隔底物到达催化性裂缝的形式阻碍底物到达活性位点所造成的结果。也有可能(至少部分上)是由于因偶联聚合分子引起的张力而使酶的3D结构发生了不利的微小结构变化所致。保持的剩余活性
本发明的多肽-聚合物偶联物显著保持了功能活性。
在本发明上下文中,“显著”保持的功能活性定义为,与在相应亲本多肽的基础上制备的偶联物活性相比,活性至少为20%-30%,优选30%-40%,更优选40%-60%,更好是从60%直至80%,还要更好是从80%直至100%。
在无聚合分子偶联至功能性位点处或者附近的本发明多肽-聚合物偶联物的情况下,剩余活性甚至可达100%或接近于此。若除去了功能性位点处的亲本肽连接基团,则其活性与修饰的(即偶联了聚合物的)亲本多肽偶联物相比,甚至可以超过100%。偶联的聚合分子的位置
为了使免疫应答(即免疫原性和变应原性应答)最佳地减小,偶联至所述多肽表面的聚合分子之间应间隔适当的距离。
在本发明的优选实施方案中,对亲本多肽进行修饰的方式能使得聚合分子广泛分布于多肽的表面上。若所述多肽具有酶促活性,则以在活性位点区域处或者附近偶联尽量少的聚合分子、尤其是不偶联聚合分子为佳。
在本发明的上下文中,“广泛分布于多肽表面”是指可用的连接基团所处的位置能使得聚合分子遮蔽表面的不同部分(优选地是远离功能性位点的整个表面或接近于整个表面),以确保表位受到遮蔽,从而不被免疫系统或其抗体所识别。
如Sheriff等(1987,美国国家科学院院报,84,p8075-8079)所述,抗体-多肽相互作用区域通常覆盖一个5002的区域。5002相当于一个25×20的长方形区或者一个半径为12.6的环形区。因此,为避免抗体与所述多肽的表位结合,以两个连接基团之间最大距离大约10为佳。
这样,所处位置距离已有的可用连接基团10以上的氨基酸残基就是合适的目标残基。如果多肽上两个或多个连接基团相距非常近,则绝大多数情形下会导致只偶联一个聚合分子。为确保功能活性损失最小,以在功能性位点处或者附近不偶联聚合分子为佳。这一距离至少部分取决于待偶联的聚合分子的庞大程度,因为并不希望由于大的聚合分子而阻碍进入功能性位点。因此,聚合分子越庞大,所偶联的聚合分子离功能性位点的距离就应越远。
为了保持目的多肽的显著功能活性,对位于距离这种功能活性位点5范围内、优选8内、特别是10范围内的连接基团应不进行偶联,因此,通过突变除去或者改变这些连接基团是有好处的。尽管功能性残基可能会是偶联聚合分子的目标,但通常不应将它们突变/除掉。因此,在这种情况下,选择涉及不同连接基团的偶联化学可能要比较好一些。
此外,按照本发明,为了提供在免疫系统可识别的已知表位或者所述表位附近偶联了聚合分子的一种多肽,在这种位点进行特定突变也被视为是有好处的。若表位的位置未知,则将数个或者许多聚合分子偶联至多肽上比较好。
正如以上所述,优选所述连接基团广泛分布于表面上。连接基团
实际上,所有的离子化基团,如赖氨酸残基的氨基,均位于多肽分子的表面上(参见例如Thomas E.Creighton,1993,“蛋白质”,W.H.Freeman and Company,New York)。
因此,修饰过的或者亲本多肽上易于达到的连接基团(如氨基)数一般等于该多肽一级结构中的赖氨酸残基数加上N-末端氨基。
将聚合分子偶联至氨基的化学比较简单,并且在本领域内已很好地建立起来了。因此,优选将赖氨酸残基(即连接基团)添加至所述亲本多肽上,和/或从所述亲本多肽上除去赖氨酸残基,以获得免疫原性和/或变应原性有所降低、并且/或者稳定性有所改善并且/或者保持的功能活性百分比比较高的改良偶联物。
也可以将聚合分子偶联至多肽表面上的氨基酸残基的羧基(-COOH)上。因此,若使用羧基(包括C-末端羧基)作为连接基团,那么,按照本发明,添加和/或除去天冬氨酸和谷氨酸残基可能也比较合适。
若使用其它的连接基团,如-SH基,则可以类似地添加和/或除去这种基团。
氨基酸残基取代优于插入,因为这样对多肽3D结构的影响通常没有那么显著。
优选的取代是保守性取代。在提高连接基团数的情形下,在距离功能性位点(酶的活性位点)5、优选8、特别是10的位置处进行取代比较有利。
旨在获得额外氨基连接基团的一个合适保守性取代的例子是精氨酸至赖氨酸的取代。旨在获得额外羧基连接基团的保守性取代的例子有:天冬酰胺至天冬氨酸/谷氨酸的取代或者谷氨酰胺至天冬氨酸/谷氨酸的取代。为了除去连接基团,可以用精氨酸等等取代赖氨酸残基。亲本多肽
在本发明上下文中,术语“多肽”包括用于医药或工业应用中的蛋白质、肽和/或酶。通常所讨论的多肽分子量约为1-100kDa,常常是15kDa-100kDa。
药用多肽
术语“药用多肽”定义为导入人体和/或动物体内的循环系统中时具有生理学活性的多肽,包括肽(如肽激素)、蛋白质和/或酶。
导入至循环系统中时,药用多肽具有潜在免疫原性。
本发明所称的“药用多肤”的例子包括胰岛素、ACTH、胰高血糖素、促生长素抑制素、促生长素、胸腺素、甲状旁腺素、色素激素(pigmentary hormones)、生长调节素、红细胞生成素、黄体生成素、绒毛膜促性腺激素、下丘脑释放因子、抗利尿素、促甲状腺素、松弛素、干扰素、血小板生成素(TPO)和催乳素。工业用多肽
工业应用中所用多肽常常具有酶促活性。工业用多肽(如酶)并不打算引入到体内的循环系统中(正好与药用多肽形成对照)。
工业用多肽,例如用作工业组合物和/或产品(如洗涤剂和个人护理产品,包括化妆品)中成分的酶,一般不太可能与人体或动物体的循环系统直接接触,因为这类酶(或包含这类酶的产品)不会被注射(或经类似方法)到血流中。
因此,在工业用多肽的情形下,潜在危险在于因通过呼吸通道吸入多肽而引发呼吸性变态反应(即IgE应答)。
在本发明上下文中,“工业用多肽”指的是并不打算导入到人体和/或动物体内循环系统中的多肽,包括肽、蛋白质和/或酶。
这类多肽的例子有诸如下述的产品中所用的多肽,特别是酶:洗涤剂、生活用产品、农业化学物质、个人护理产品(如皮肤护理产品,包括化妆品和洗涤用品)、口腔和皮肤药物、用于加工纺织品的组合物、用于硬表面清洁的组合物,以及用于生产食品和饲料的组合物,等等。酶促活性
具有酶促活性的药用或者工业用多肽常常属于包括下述的酶类之一:氧化还原酶(E.C.1,“酶命名法,(1992),学院出版公司),如漆酶和超氧化物歧化酶(SOD);转移酶,(E.C.2),如转谷氨酰胺酶(TG酶);水解酶(E.C.3),包括蛋白酶,特别是枯草杆菌蛋白酶,以及脂解酶;异构酶(E.C.5),如蛋白质二硫键异构酶(PDI)。
水解酶
蛋白水解酶
所涉及的蛋白水解酶包括选自下列组的蛋白酶:天冬氨酸蛋白酶(如胃蛋白酶)、半胱氨酸蛋白酶(如木瓜蛋白酶)、丝氨酸蛋白酶(如枯草杆菌蛋白酶),或金属蛋白酶(如Neutrase)。
亲本蛋白酶的一些具体例子包括:PD498(WO 93/24623,SEQ.IDNO.2),Savinase(von der Osten等,(1993),生物技术杂志,28,p.55+,SEQ ID NO:3),蛋白酶K(Gunkel等,(1989),欧洲生物化学杂志,179,p.185-194),蛋白酶R(Samal等,(1990),分子微生物学,4,p.1789-1792),蛋白酶T(Samal等,(1989),基因,85,P.329-333),枯草杆菌蛋白酶DY(Betzel等,(1993),Arch.Biophys,302,no.2,P.499-50),Lion Y(JP 04197182-A),Rennilase(可从Novo NordiskA/S得到),JA16(WO 92/17576),Alcalase(天然枯草杆菌蛋白酶Carlberg变体)(von der Osten等,(1993),生物技术杂志,28,P.55+)
脂解酶
所涉及的脂解酶包括柔毛腐质霉(Humicola lanuginosa)脂肪酶(如在EP 258 068和EP 305 216中描述的那一种,参见下述SEQ IDNO:6),Humicola insolens、Rhizomucor miehei脂肪酶(如EP 238023所述),犁头霉属之种的脂解酶(WO 96/13578),假丝酵母属脂肪酶(如C.antarctica脂肪酶,例如在EP 214 761中描述的C.antarctica脂肪酶A或B),假单胞菌属脂肪酶(例如产碱假单胞菌、类产碱假单胞菌脂肪酶,如EP 218 272中所述;洋葱假单孢菌脂肪酶,如EP 331376中所述;WO95/14783中所公开的假单孢菌脂肪酶),芽孢杆菌脂肪酶[例如枯草芽孢杆菌脂肪酶(Dartois等,(1993)Biochemica etBiophysica acta,1131,253-260)、嗜热脂肪芽孢杆菌脂肪酶(JP64/744992)和短小芽孢杆菌脂肪酶(WO 91/16422)]。其它类型的脂解酶包括角质酶,例如在WO 88/09367中描述的源自门多萨假单胞菌的角质酶,或源自Fusarium solani pisi的角质酶(如在WO 90/09446中所述)。
氧化还原酶
漆酶
所涉及的漆酶包括Polyporus pinisitus漆酶(WO 96/00290),毁丝霉属漆酶(WO 95/33836),Schytalidium属漆酶(WO95/338337),以及Pyricularia oryzae漆酶(可得自Sigma)。
过氧化物酶
所涉及的过氧化物酶包括短小芽孢杆菌过氧化物酶(WO91/05858),粘球菌科过氧化物酶(WO95/11964),灰盖鬼伞过氧化物酶(WO95/10602),以及Arrthromyces ramosus过氧化物酶[Kunishima等(1994),分子生物学杂志,235,331-344]。
转移酶
转谷氨酰胺酶
合适的转移酶包括在WO 96/06931(Novo Nordisk A/S)和WO96/22366(Novo Nordisk A/S)中所公开的任何转谷氨酰胺酶。
异构酶
蛋白质二硫键异构酶
合适的蛋白质二硫键异构酶包括在WO 95/01425(Novo NordiskA/S)中描述的那些PDI,但并不限于此。
聚合分子
偶联到多肽上的聚合分子可以是任何合适的聚合分子,包括天然和合成的同聚物[如多元醇(即poly-OH)、聚胺(即poly-NH2)以及聚羧酸(即poly-COOH)]以及杂聚物,即包含一种或多种不同偶联基团(如羟基与胺基)的聚合物。
合适的聚合分子的例子包括选自含有下述各类分子的组中的聚合分子:聚烯化氧(PAO),如聚亚烷基二醇(PAG),包括聚乙二醇(PEG),甲氧基聚乙二醇(mPEG)以及聚丙二醇、PEG-缩水甘油醚(Epox-PEG)、PEG-氧羰基咪唑(CDI-PEG)、分支PEG、聚乙烯醇(PVA)、聚羧酸、聚(乙烯吡咯烷酮)、聚D,L-氨基酸、乙烯-马来酸酐共聚物、苯乙烯-马来酸酐共聚物,葡聚糖,包括羧甲基葡聚糖、肝素、同源的白蛋白、纤维素,包括甲基纤维素、羧甲基纤维素、乙基纤维素、羟乙基纤维素、羧乙基纤维素以及羟丙基纤维素,聚氨基葡糖的水解产物、淀粉(如羟乙基淀粉和羟丙基淀粉)、糖原、琼脂糖及其衍生物、瓜尔胶、支链淀粉、菊粉、合成生物聚合胶(xanthan gum)、角叉菜胶、果胶、藻酸水解产物和生物聚合物。
优选的聚合分子是无毒性的聚合分子,如(m)聚乙二醇((m)PEG),还要求这些分子共价偶联到酶表面连接基团上的化学方法相对比较简单。
一般看来,聚烯化氧(PAO)(如聚氧化乙烯,如PEG,特别是mPEG)是优选的聚合分子,因为这些聚合分子与多糖(如葡聚糖、支链淀粉等)相比,它们所具有的能交联的活性基团很少。
按照本发明,尽管可以使用所有上述的聚合分子,但优选地使用甲氧基聚乙二醇(mPEG)。这是因为这样的事实:甲氧基乙二醇仅具有一个可与酶偶联的活性末端。因而,交联的可能性相对较不明显。而且,这将使产物更均一,聚合分子与酶的反应更容易控制。制备酶变体
可以通过任何适当方法构建待偶联的酶变体。本领域内已成功建立了一些方法。例如,可以利用下述文献中的相同材料和方法制备本发明的酶变体:WO89/06279(Novo Nordisk A/S),EP130,756(Genentech),EP479,870(Novo Nordisk A/S),EP214,435(Henkel),WO87/04461(Amgen),WO87/05050(Genex),EP申请号87303761(Genentech),EP260,105(Genencor),WO88/06624(Gist-Brocades NV),WO88/07578(Genentech),WO88/08028(Genex),WO88/08033(Amgen),WO88/08164(Genex),Thomas等(1985)自然,318,375-376;Thomas等(1987)分子生物学杂志,193,803-813;Russel和Fersht(1987)自然,328,496-500。产生定点突变
诱变前,必须将编码目的多肤的基因克隆到合适的载体中。下面将介绍在特定位点产生突变的方法。
一旦克隆到了多肽编码基因、鉴定出了进行突变的理想位点,并且决定了用何种残基取代原残基,则可以利用合成寡核苷酸导入这些突变。这些寡核苷酸含有目的突变位点旁侧的核苷酸序列,并且在寡核苷酸合成中插入了突变核苷酸。在一种优选方法中,定点诱变是通过Kammann等[(1989)核酸研究,17(13),5404]和Sarkar G.和Sommer,S.S.[(1990),生物技术,8,404-407]所述的SOE-PCR诱变技术进行的。
聚合物的活化
如果将与多肽偶联的聚合分子没有活性,就必须使用合适方法使其活化。根据本发明,可以想见,可通过接头将聚合分子与多肽偶联。合适的接头对熟练技术人员是熟知的。
用于活化聚合分子以及用于偶联多肽的方法和化学在文献中有详细描述。一般用于活化不溶性聚合物的方法包括:用下列化合物活化官能团:溴化氰、高碘酸、戊二醛、biepoxides、表氯醇、二乙烯基砜、碳二亚胺、磺酰基卤化物、三氯三嗪等(参见R.F.Taylor,(1991),“蛋白质固定,基础和应用”,Marcel Dekker,N.Y.;S.S.Wong,(1992),“蛋白质结合和交联的化学”,CRC出版社,BocaRaton;G.T.Hermanson等,(1993),“固定的亲和性配体技术”,学院出版社,N.Y.)。其中一些方法涉及不溶性聚合物的活化,但是也适用于可溶性聚合物的活化,如高碘酸,三氯三嗪、磺酰基卤化物、二乙烯基砜、碳二亚胺等。在选择活化与偶联化学方法时,必须考虑聚合物上的官能团是氨基、羟基、巯基、羧基、醛还是硫氢基以及蛋白质上选定的连接基团,活化与偶联化学通常包括:i)聚合物的活化,ii)偶联,iii)剩余活性基团的封闭。
下面简要描述许多合适的聚合物活化方法。然而,应该理解也可以使用其它方法。
将聚合分子偶联到多肽的游离酸基团上可以借助于二酰亚胺和,例如氨基-PEG或肼基-PEG(Pollak等,(1976),J.Amr.Chem.Soc.,98,289-291)或重氮基乙酸酯/酰胺(Wong等,(1992),“蛋白质结合和交联化学”,CRC出版社)进行。
将聚合分子偶联到羟基通常是非常困难的,因为这必须在水中进行。而通常水解反应比与羟基的反应更占优势。
将聚合分子偶联到游离硫氢基可以用特定基团(如马来酰亚胺或正吡啶基二硫化物)达到。乙烯基砜(美国专利no.5,414,135,(1995),Snow等)对于硫氢基也有偏好,但是不象提到的其它化合物那样有选择性。
可以通过包含两个邻近羰基的基团靶向多肽链中的可及精氨酸残基。
涉及将亲电子性地活化的PEG偶联到赖氨酸的氨基上的技术也有用。醇的许多常规离去基团能形成胺键。例如,可以使用烷基磺酸盐,如tresylates(Nilsson等,(1984),酶学方法,104卷,Jacoby W.B.编,学院出版社:Orlando,P.56-66;Nilsson等,(1987),酶学方法,135卷,Mosbach K.编,学院出版社:Orlando,P.65-79;Scouten等,(1987),酶学方法,135卷,Mosbach K.编,学院出版社:Orlando,P.79-84;Crossland等,(1971),J.Amr.Chem.Soc.1971,93,pp.4217-4219)、甲磺酸盐(Harris,1985,同上;Harris等,(1984),J.Polym.Sci.Polym.Chem.Ed.22,pp 341-352)、芳基磺酸盐(如甲苯磺酸盐)以及对硝基苯磺酸盐。
有机磺酰氯(例如Tresyl Chloride)可有效地将许多聚合物(例如PEG)中的羟基转化成良好的离去基团(磺酸盐),当与多肽中的亲核基团(如氨基)进行反应时,该离去基团使得可以在聚合物和多肽之间形成稳定的键。除了要有高偶联产率之外,反应条件通常还要求比较温和(中性或微碱性pH值,以避免变性,且活性极少或没有破坏),并且满足多肽需要的非破坏性条件。
甲苯磺酸盐比甲磺酸盐反应性更强,而且更不稳定地分解成PEG、二噁烷和磺酸(Zalipsky,(1995),生物结合物化学,6,150-165)。环氧衍生物也可以用于产生胺键,但是活性比上述基团弱得多。
用碳酰氯将PEG转化成氯甲酸酯将产生与赖氨酸的氨基甲酸酯键。这一转变可以多种变化形式进行,如用N-羟基琥珀酰亚胺(美国专利no.5,122,614,(1992);Zalipsky等,(1992),生物技术应用和生物化学,15,P.100-114;Monfardini等,(1995),生物结合物化学,6,62-69)、用咪唑(Allen等,(1991),Carbohydr.Res.,213,pp 309-319)、用对硝基苯酚、DMAP(EP 632 082 A1,(1993),Looze,Y.)等取代氯。通常通过使氯甲酸酯与所需的离去基团反应来制备衍生物。所有这些基团均产生与肽的氨基甲酸酯键。
此外,可以分别使用异氰酸盐和异硫氰酸盐来产生脲和硫脲。
使用如上所述的相同的离去基团和cyclic imid thrones,可以从PEG酸获得酰胺(美国专利no.5,349,001,(1994),Greenwald等)。这些化合物的反应活性十分高,但是可能会使水解变快。
也可以使用从与琥珀酸酐反应制备的PEG琥珀酸酯。由此所形成的酯基使偶联物对水解更加敏感得多(美国专利no.5,122,614,(1992),Zalipsky)。这一基团可以用N-羟基琥珀酰亚胺活化。
此外,可以引入一个特殊的接头。最古老的是氰尿酰氯(Abuchowski等,(1977),生物化学杂志,252,3578-3581;美国专利no.4,179,337,(1979),Davis等;Shafer等,(1986),J.Polym.Sci.Polym.Chem.Ed.,24,375-378)。
将PEG偶联到芳香族胺上,随后重氮化,将产生非常活跃的重氮盐,其在原位即可与肽反应。通过使PEG的吖内酯衍生物(美国专利no.5,321,095,(1994),Greenwald,R.B.)反应,也可以形成酰胺键,这样即可引入一个额外的酰胺键。
由于一些肽不包含许多赖氨酸,因此,将一个以上的PEG连接到同一赖氨酸上可能会是有利的。这可以通过例如使用1,3-二氨基-2-丙醇进行。
也可以通过氨基甲酸酯键将PEG连接到酶的氨基上(WO 95/11924,Greenwald等)。赖氨酸残基也可以用作主链。
实施例中所用的偶联技术是WO90/13590(Enzon)中所述的琥珀酰亚胺碳酸酯偶联技术。制备改良偶联物的方法
本发明的又一目的是提供制备改良的多肽-聚合物偶联物的方法,包括以下步骤:a)鉴定出位于所讨论的亲本多肽3D结构表面上的氨基酸残基,b)选择出所述亲本多肽的所述3D结构表面上欲进行突变的目标氨基酸残基,c)i)用具有合适连接基团的氨基酸残基取代步骤b)中选定的一个或多个氨基酸残基或者在所选位点插入所述氨基酸残基,和/或
ii)取代或者删除步骤b)中选定的、功能性位点处或者附近的一个或多个氨基酸残基,d)将聚合分子偶联到已突变的多肽上。步骤a)鉴定出位于亲本多肽表面上的氨基酸残基三维结构(3D-结构)
为了进行本发明方法,需要有所述亲本多肽的三维结构。这一结构例如可以是X-射线结构、核磁共振结构或建立的模型结构。Brookhaven数据库可以作为X-射线结构和核磁共振结构的来源。
如果与所说的多肽有至少30%序列等同的一种或多种同源多肽的一个或多个3D结构已知,则可由本领域技术人员建立模型结构。有几个软件包可以用来构建模型结构,一个例子是Biosym的Homology95.0软件包。
构建模型结构所需要的典型工作是:对已知3D结构的同源序列进行序列对比、确定结构保守区域(SCR)、确定SCR的等同序列、在结构数据库中搜索结构片段/环以取代变异区、确定这些区域的等同序列、通过能量最低化使结构精确化。已知相对于已知的3D结构含有大插入物(≥3个残基)的区域相当难以模拟,必须小心地预测其结构。
获得所说多肽的3D-结构或基于与已知结构的同源性建立的结构模型后,这种结构即可作为必要先决条件用于完成下述方法。步骤b)选出用于突变的目标氨基酸残基
根据本发明,选出欲进行突变的目标氨基酸残基,以在多肽表面获得额外或者更少的连接基团,如游离氨基(-NH2)或游离羧酸基团(-COOH),和/或使多肽表面上的表位更为完全和广泛地受到遮蔽。保守性取代
优选在多肽内进行保守性取代,因为保守性取代比较安全,突变对多肽结构的影响有限。
为了提供额外氨基,可以用赖氨酸取代精氨酸,这两种残基都带正电,但仅赖氨酸具有适于用作连接基团的游离氨基。
为提供额外羧酸基团,保守性取代可以是天冬酰胺至天冬氨酸的取代或者谷氨酰胺至谷氨酸的取代。这些残基在大小、形状方面比较相象,只是这种羧酸基团只存在于酸性残基上。
为了减少诸如活性位点处或者附近的连接基团,可以用精氨酸残基取代赖氨酸,等等。
用何种氨基酸进行取代在原则上取决于将要应用的偶联化学。非保守性取代
突变也可以对不太/不保守的目标氨基酸残基进行。为了对多肽表面进行比保守性取代所能达到的遮蔽程度更为完全和广泛的遮蔽,这种突变就比较合适。
先用普通术语描述本发明方法,然后再用具体实例进行描述。
注意以下术语的用法:
连接残基:可结合聚合分子的残基,如赖氨酸(氨基)或天冬氨酸/谷氨酸(羧基)。也包括相关的N-或C-末端氨基/羧基。
突变残基:待突变的残基,如精氨酸或者天冬酰胺/谷氨酰胺。
必需的催化性残基:已知催化功能所必需的残基,如丝氨酸蛋白酶内的催化性三联体。
溶剂暴露型残基定义为,按照Homology 95.0模块内的BIOSYM/INSIGHT算法,至少暴露5%的残基。各指令的顺序如下:HomologyProstatAccess_Surf=>Solv_Radins 1.4;Heavy atomsonly;Radii source VdW;O utput:Fractional Area;Polaritysource:Defaul。得到文件名为area.tab的文件。备注:为使该程序适当地运行,必须首先将结构中的所有水分子完全除去。
例如,看起来可以是这样的:# PD498FINALMODEL# 残基 面积TRP_1 136.275711SER_2 88.188095PRO_3 15.458788ASN_4 95.322319ASP_5 4.903404PRO_6 68.096909TYR_7 93.333252TYR_8 31.791576SER_9 95.983139-- 续
1.鉴定出距离最近的连接残基10以上、所处位置距离必需的催化性残基至少8的诸残基。这一残基亚组称作REST,是进行由突变残基至连接残基保守性取代的主要区域。
2.鉴定出位于REST亚组周围0-5的壳内、但距离必需的催化性残基至少8的残基。这一残基亚组称作SUB5B。这是进行由突变残基至连接残基保守性取代的次要区域。因为结合至SUB5B内连接残基上的配体会延伸到REST区内,有可能会阻止表位识别。
3.鉴定出REST和SUB5B内的溶剂暴露型突变残基,作为导入连接残基的潜在突变位点。
4.利用BIOSYM/INSIGHT的生物聚合物模块,取代操作3中鉴定出的残基。
5.重复上述第1-2项操作,得到RESTX亚组。这一亚组包括距离最近的连接残基至少10、所处位置远离必需的催化性残基至少8的那些残基。
6.鉴定出RESTX内的溶剂暴露型残基。它们是进行不太/不保守的突变以导入连接残基的潜在位点。步骤c)取代、插入或删除氨基酸残基
可以利用本领域众所周知的标准技术,如定点诱变(参见例如Sambrook等(1989),Sambook等,分子克隆;实验手册,冷泉港,NY),进行步骤c)中讨论的突变。
核苷酸取代的一般性描述可见于例如Ford等,1991,蛋白质表达和纯化,2,第95-107页。步骤d)将聚合分子偶联至经修饰的亲本酶上
可以通过本领域已知的任何偶联方法,包括上述技术,制备本发明的多肽-聚合物偶联物。
将聚合分子偶联到所讨论的多肽上
如果将与多肽偶联的聚合分子没有活性,就必须使用合适方法使其活化。聚合分子可通过接头与多肽偶联。合适的接头对熟练技术人员是熟知的。
用于活化聚合分子以及用于偶联多肽的方法和化学在文献中有详细描述。一般用于活化不溶性聚合物的方法包括:用下列化合物活化官能团:溴化氰、高碘酸、戊二醛、biepoxides、表氯醇、二乙烯基砜、碳二亚胺、磺酰基卤化物、三氯三嗪等(参见R.F.Taylor,(1991),“蛋白质固定,基础和应用”,Marcel Dekker,N.Y.;S.S.Wong,(1992),“蛋白质结合和交联的化学”,CRC出版社,BocaRaton;G.T.Hermanson等,(1993),“固定的亲和性配体技术”,学院出版社,N.Y.)。其中一些方法涉及不溶性聚合物的活化,但是也适用于可溶性聚合物的活化,如高碘酸、三氯三嗪、磺酰基卤化物、二乙烯基砜、碳二亚胺等。在选择活化与偶联化学方法时,必须考虑聚合物上的官能团是氨基、羟基、巯基、羧基、醛还是硫氢基以及蛋白质上选定的连接基团,活化与偶联化学通常包括:i)聚合物的活化,ii)偶联,iii)剩余活性基团的封闭。
下面简要描述许多合适的聚合物活化方法。然而,应该理解也可以使用其它方法。
将聚合分子偶联到酶的游离酸基团上可以借助于二酰亚胺和,例如氨基-PEG或肼基-PEG(Pollak等,(1976),J.Amr.Chem.Soc.,98,289-291)或重氮基乙酸酯/酰胺(Wong等,(1992),“蛋白质结合和交联化学”,CRC出版社)进行。
将聚合分子偶联到羟基通常是非常困难的,因为这必须在水中进行。而通常水解反应比与羟基的反应更占优势。
将聚合分子偶联到游离硫氢基可以用特定基团(如马来酰亚胺或正吡啶基二硫化物)达到。乙烯基砜(美国专利no.5,414,135,(1995),Snow等)对于硫氢基也有偏好,但是不象提到的其它化合物那样有选择性。
可以通过包含两个邻近羰基的基团靶向多肽链中的可及精氨酸残基。
涉及将亲电子性地活化的PEG偶联到赖氨酸的氨基上的技术也有用。醇的许多常规离去基团能形成胺键。例如,可以使用烷基磺酸盐,如tresylates(Nilsson等,(1984),酶学方法,104卷,Jacoby W.B.编,学院出版社:Orlando,P.56-66;Nilsson等,(1987),酶学方法,135卷,Mosbach K.编,学院出版社:Orlando,P.65-79;Scouten等,(1987),酶学方法,135卷,Mosbach K.编,学院出版社:Orlando,P.79-84;Crossland等,(1971),J.Amr.Chem.Soc.1971,93,pp.4217-4219)、甲磺酸盐(Harris,1985,同上;Harris等,(1984),J.Polym.Sci.Polym.Chem.Ed.22,pp 341-352)、芳基磺酸盐(如甲苯磺酸盐)以及对硝基苯磺酸盐。
有机磺酰氯(例如Tresyl Chloride)可有效地将许多聚合物(例如PEG)中的羟基转化成良好的离去基团(磺酸盐),当与多肽中的亲核基团(如氨基)进行反应时,该离去基团使得可以在聚合物和多肽之间形成稳定的键。除了要有高偶联产率之外,反应条件通常还要求比较温和(中性或微碱性pH值,以避免变性,且活性极少或没有破坏),并且满足多肽需要的非破坏性条件。
甲苯磺酸盐比甲磺酸盐反应性更强,而且更不稳定地分解成PEG、二噁烷和磺酸(Zalipsky,(1995),生物结合物化学,6,150-165)。环氧衍生物也可以用于产生胺键,但是活性比上述基团弱得多。
用碳酰氯将PEG转化成氯甲酸酯将产生与赖氨酸的氨基甲酸酯键。这一转变可以多种变化形式进行,如用N-羟基琥珀酰亚胺(美国专利no.5,122,614,(1992);Zalipsky等,(1992),生物技术应用和生物化学,15,P.100-114;Monfardini等,(1995),生物结合物化学,6,62-69)、用咪唑(Allen等,(1991),Carbohydr.Res.,213,pp 309-319)、用对硝基苯酚、DMAP(EP 632 082 A1,(1993),Looze,Y.)等取代氯。通常通过使氯甲酸酯与所需的离去基团反应来制备衍生物。所有这些基团均产生与肽的氨基甲酸酯键。
此外,可以分别使用异氰酸盐和异硫氰酸盐来产生脲和硫脲。
使用如上所述的相同的离去基团和cyclic imid thrones,可以从PEG酸获得酰胺(美国专利no.5,349,001,(1994),Greenwald等)。这些化合物的反应活性十分高,但是可能会使水解变快。
也可以使用从与琥珀酸酐反应制备的PEG琥珀酸酯。由此所形成的酯基使偶联物对水解更加敏感得多(美国专利no.5,122,614,(1992),Zalipsky)。这一基团可以用N-羟基琥珀酰亚胺活化。
此外,可以引入一个特殊的接头。最古老的是氰尿酰氯(Abuchowski等,(1977),生物化学杂志,252,3578-3581;美国专利no.4,179,337,(1979),Davis等;Shafer等,(1986),J.Polym.Sci.Polym.Chem.Ed.,24,375-378)。
将PEG偶联到芳香族胺上,随后重氮化,将产生非常活跃的重氮盐,其在原位即可与肽反应。通过使PEG的吖内酯衍生物(美国专利no.5,321,095,(1994),Greenwald,R.B.)反应,也可以形成酰胺键,这样即可引入一个额外的酰胺键。
由于一些肽不包含许多赖氨酸,因此,将一个以上的PEG连接到同一赖氨酸上可能会是有利的。这可以通过例如使用1,3-二氨基-2-丙醇进行。
也可以通过氨基甲酸酯键将PEG连接到酶的氨基上(WO 95/11924,Greenwald等)。赖氨酸残基也可以用作主链。连接基团的添加PD498变体-SPEG偶联物的具体例子
蛋白酶的一个具体例子是亲本PD498(WO 93/24623,SEQ ID NO.2)。亲本PD498分子量为29kDa。
赖氨酸和精氨酸位置如下所示:
离活性位点的距离 精氨酸 赖氨酸
0-5 1
5-10
10-15 5 6
15-20 2 3
20-25 1 3
总共 9 12
发明者观察了亲本PD498表面上的哪些位点适于导入额外的连接基团。
A.亲本PD498中合适的精氨酸至赖氨酸保守性取代可以是R51K,R62K,R121K,R169K,R250K,R28K,R190K中的任一种。
B.亲本PD498中合适的非保守性取代可以是以下取代中的任一种:P6K,Y7K,S9K,A10K,Y11K,Q12K,D43K,Y44K,N45K,N65K,G87K,I88K,N209K,A211K,N216K,N217K,G218K,Y219K,S220K,Y221K,G262K。
因为没有赖氨酸残基位于活性位点处或附近,所以无需去除任何连接基团。
可通过本领域已知的将聚合分子偶联到酶上氨基基团的任何偶联技术,用前面提到的任一种PD498变体作为起始材料制备PD498变体-SPEG偶联物。下面介绍一个具体例子。连接基团的去除BPN’变体-SPEG偶联物的具体例子
在活性位点有连接基团的蛋白酶的一个具体例子是BPN’,BPN’有11个连接基团(加上一个N-末端氨基基团),它的分子量为28kDa。赖氨酸和精氨酸残基位置如下:
距活性位点的距离 精氨酸 赖氨酸
0-5 1
5-10
10-15 1 4
15-20 1 4
20-25 2
总共 2 11
按照本发明,除去位于距活性位点0-5之内的赖氨酸残基比较有利。具体地说,这可通过K94R取代来完成。
用上述BPN’变体作为起始材料,使用本领域已知的将聚合分子偶联至酶上氨基基团的任何偶联技术,可制备BPN’变体-SPEG偶联物。连接基团的添加和去除Savinase-SPEG偶联物的具体例子
如实施例2中所说,按照本发明,可以在亲本Savinase(Von derOsten等人.,(1993),生物技术杂志,28,p.55+,SEQ ID NO.3)表面上添加数个氨基连接基团,并去除靠近其活性位点的氨基连接基团。
亲本Savinase内的下述取代中的任一种都是诱变的位点:R10K,R19K,R45K,R145K,R170K,R186K和R247K。
K94R取代被验明为一适于阻止聚合物连接至活性位点附近的突变。
用任何上述Savinase变体作为起始材料,通过使用本领域中已知的用于偶联聚合分子至酶上氨基基团的任何偶联技术,可制备Savinase变体-SEPG偶联物。连接基团的添加Humicola lanuginosa脂肪酶变体-SPEG偶联物的具体例子
将亲本Humicola lanuginosa DSM 4109脂肪酶(见SEQ ID NO.6)作为主链进行取代、免疫原性有所降低的脂肪酶变体具体例子列举如下。
未修饰的亲本Humicola lanuginosa脂肪酶有8个连接基团,包括N-末端NH2基团,它的分子量约为29kDa。A.亲本脂肪酶中,合适的精氨酸到赖氨酸保守性取代可以是以下的任一种:R133K、R139K、R160K、R179K、R209K、R118K及R125K。
亲本脂肪酶中合适的非保守性取代可为以下任一种:A18K、G31K、T32K、N33K、G38K、A40K、D48K、T50K、E56K、D57K、S58K、G59K、V60K、G61K、D62K、T64K、L78K、N88K、G91K、N92K、L93K、S105K、G106K、V120K、P136K、G225K、L227K、V228K、P229K、P250K、F262K。
Humicola lanuginosa脂肪酶中合适的其它非保守性取代包括:E87K或D254K。
以上述脂肪酶变体中任一种作为起始材料,用本领域已知用于偶联聚合分子至酶上氨基基团的任何偶联技术,可制备脂肪酶变体-SPEG偶联物。下面介绍一个具体例子。
下面的实施例12显示,与相应的未修饰亲本酶相比,具有E87K+D254K取代并偶联了S-PEG15,000的Humicola lanuginosa脂肪酶变体偶联物在Balb/c小鼠中的免疫应答有所降低。免疫原性和变应原性
免疫原性是一个比“抗原性”和“变应原性”含义更广的词,表示免疫系统对外来物质出现产生的应答。根据诱发的免疫应答的类型,将所说的外来物质称为免疫原、抗原和变应原。
“免疫原”可定义为,当导入动物和人的循环系统时,能激发免疫应答、导致免疫球蛋白形成的物质。
“抗原”一词指,当被识别为异已分子时,其本身即能产生抗体的物质。
另外,“变应原”则可定义为,能通过IgE抗体(人类中,以及动物中具相似效果的分子)引起变应性致敏或变应性应答的抗原。免疫原性的评估
皮下注射动物,使免疫原进入循环系统,将其应答与对相应亲本多肽的应答进行比较,即可对免疫原性进行评估。
在本发明的上下文中,人体和动物体的“循环系统”指主要包括心脏和血管的系统。心脏运送必需的能量以保持血管系统中的血液循环。循环系统起着生物体传送系统的功能,血液运送O2、营养物质、激素及对细胞调控比较重要的其它物质至组织中。此外,血液将二氧化碳从组织中排出至肺,将残留物质排出至例如肾中。而且,血液对温度调节及身体的防卫机制(包括免疫系统)具重要作用。
有许多体外动物模型可用于评估多肽的免疫原性潜能。其中一些模型为人体中的危害评估提供了合适的基础。合适的模型包括小鼠模型。
这一模型力求鉴别皮下注射了被修饰和未修饰多肽的Balb/c小鼠中以IgG应答形式表现的免疫原性应答。
还有其它的动物模型可用于免疫原性潜能的评估。
按照本发明,具有“降低的免疫原性”的多肽即指与相应的亲本多肽相比,所述多肽在导入循环系统时,产生的可引起免疫应答的抗体量明显降低,所说的抗体例如是人类的免疫球蛋白及特定动物中具类似作用的分子。
对Balb/c小鼠来说,IgG应答是多肽免疫原性潜能的一个很好的指征。变应原性的评估
变应原性的评估可通过吸入试验进行,即比较亲本酶和本发明相应的被修饰酶施用于气管内后产生的效果。
有一些体内动物模型可用于评估酶的变应原性。其中一些模型为人类中的危害评估提供了合适的基础。合适的模型包括豚鼠模型和小鼠模型。这些模型力求将呼吸性变应原鉴定为在事先致敏的动物中引发刺激反应的函数。根据这些模型,将预计的变应原气管内导入动物中。
豚鼠的一合适品系,Dunkin Hartley品系,不象人类那样产生与变应性应答有关的IgE抗体。可是,它们产生其它类型的抗体-IgG1A和IgG1B(参见例如Prent,ATLA,19,p.8-14,1991),这些抗体负责对包括酶在内的吸入多肽产生的变应原性应答。因此,在使用Dunkin Hartley动物模型的时候,IgG1A和IgG1B的相对量是变应原性水平的一个衡量尺度。
Balb/c品系小鼠适于进行气管内暴露。Balb/c小鼠产生IgE作为变应性应答。
有关豚鼠和小鼠中呼吸性变应原的评估的更多细节描述于Kimber等人(1996年),《基础和应用毒理学》,33卷,第1-10页。
其它的动物,如大鼠、兔子等,也可用于类似的研究中。组合物
本发明涉及包含本发明中的多肽-聚合物偶联物的组合物。
该组合物可以是药用或工业用组合物。
该组合物可进一步包含其它多肽、蛋白质或酶及/或常用于诸如洗涤剂中的成分,包括肥皂、家用物品、农业化肥,个人护理产品,包括皮肤护理组合物、清洁用组合物(如用于接触性镜片)、口腔和皮肤药用品,还有用于处理纺织品的组合物,用于生产食品(如烘烤)和饲料的组合物,等等。多肽-聚合物偶联物的用途
本发明还涉及本发明方法用于降低多肽免疫应答的用途。
本发明的又一目的是利用本发明的多肽-聚合物偶联物来降低工业产品的变应原性,所述工业产品例如是洗涤剂(诸如洗衣、洗盘子及清洗硬表面所用的洗涤剂),和食品或饲料产品。材料与方法材料酶:PD498:枯草杆菌蛋白酶类蛋白酶,可见于WO 93/24623。PD498的序列可见于SEQ ID NO.1和2中。Savinase(来自Novo Nordisk A/S)Humicola lanuginosa脂肪酶:可以Lipolase从Novo Nordisk获得,其进一步描述于EP 305,216。其DNA和蛋白质序列分别见于SEQ ID NO.5和6中。菌株:
枯草芽孢杆菌309和147是迟缓芽孢杆菌的变体,保藏于NCIB,保藏号为NCIB10309和10147,这两个菌株描述于美国专利号3723250中,其公开内容引入本文作为参考文献。
通过常规方法将大肠杆菌MC1000(M.J.Casadaban和S.N.Cohen(1980);分子生物学杂志,138卷,179-207页)制成r-、m+,该菌株还描述于美国专利申请系列号039,298中。载体:
pPD498:包含编码PD498蛋白酶(SEQ ID NO.2)野生型基因的大肠杆菌-枯草芽孢杆菌穿梭载体(描述于美国专利号5621089中的6.2.1.6部分)。同一载体可用于在大肠杆菌中进行诱变及在枯草芽孢杆菌中进行表达。一般分子生物学方法:
除非另外指出,否则DNA操作和转化按分子生物学的标准方法进行(Sambrook等人,(1989)分子克隆:实验室手册,冷泉港实验室,冷泉港,纽约;Ausubel,F.M.等人(编),“现代分子生物学方法”,JohnWiley和Sons,1995;Harwood,C.R.和Cutting,S.M.(编)《用于芽孢杆菌的分子生物学方法》,John Wiley和Sons,1990)。
DNA操作中的酶按照产品说明书使用。
材料、化学药品和溶液:标记了辣根过氧化物酶的抗大鼠免疫球蛋白(Dako,DK,P162,# 031;稀释度1∶1000)。小鼠抗大鼠IgE(Serotec MCA 193;稀释度1∶200)。大鼠抗小鼠IgE(Serotec MCA 419;稀释度1∶100)。生物素标记的小鼠抗大鼠IgG1单克隆抗体(Zymed 039140;稀释度1∶1000)。生物素标记的大鼠抗小鼠IgG1单克隆抗体(Serotec MCA 336B;稀释度1∶1000)链霉抗生物素-辣根过氧化物酶(Kirkegard和Perry14-30-00;稀释度1∶1000)Covalink NH2平板(Nunc,目录号459439)氰尿酰氯(Aldrich)丙酮(Merck)大鼠抗小鼠IgG1,生物素(SeroTec.目录号MCA 336B)链霉抗生物素、过氧化物酶(KPL)邻苯二胺(OPD)(Kem-en-Tec)过氧化氢,30%(Merck)吐温20(Merck)脱脂奶粉(Difco)硫酸(Merck)缓冲液和溶液:碳酸盐缓冲液(0.1M,pH10(1升))碳酸钠10.60克PBS(pH7.2(1升)) 氯化钠 8.00克
氯化钾 0.20克
磷酸氢二钾 1.04克
磷酸二氢钾 0.32g洗涤缓冲液:PBS,0.05%(v/v)吐温20封闭缓冲液:PBS,2%(wt/v)脱脂奶粉稀释缓冲液:PBS,0.05%(v/v)吐温20,0.5%(wt/v)脱脂奶粉柠檬酸盐缓冲液(0.1M,pH5.0-5.2(1升)) 柠檬酸钠 20.60克
柠檬酸 6.30克Covalink平板的活化:·配制每毫升丙酮含10mg氰尿酰氯的新鲜母液。·使用前,过搅拌边将氰尿酰氯母液稀释到PBS中至终浓度1mg/ml。·加100ml稀释液至Covalink氨基平板的每一孔中,室温温育5分钟。·用PBS洗3次。·在50℃烘干新制备的活化平板30分钟。·立即用封条将每一平板密封好。·预先活化的平板放在塑料袋中可于室温保存3周。硼酸钠、硼砂(Sigma)3,3-二甲基戊二酸(Sigma)氯化钙(Sigma)Tresyl Chloride(2,2,2-三氟乙磺酰氯)(Fluka)1-乙基-3-(3-二甲基氨基丙基)碳二亚胺(EDC)(Fluka)N-羟基琥珀酰亚胺(Fluka art.56480)碳酰氯(Fluka art.79380)乳糖(Merck 7656)PMSF(苯基甲基磺酰氟)来自Sigma琥珀酰-丙氨酰-丙氨酰-脯氨酰-苯丙氨酰基对硝基苯胺(Suc-AAPF-pNP)Sigma号S-7388,分子量624.6克/摩尔。生色底物:OPD:邻苯二胺(Kementec目录号4260)试验动物:Dunkin Hartley豚鼠(来自Charles River,DE)雌性Balb/c小鼠(约20克)购自Bomholdtgaard,Ry,丹麦。设备:SCEL II(Novex)ELISA读数仪(UVmax,Molecular Devices)HPLC(Waters)PFLC(Pharmacia)Superdex-75柱,Mono-Q,Mono S,来自Pharmacia,SW。SLT:Fotometer,来自SLT LabInstruments大小排阻层析(Spherogel TSK-G 2000 SW)大小排阻层析(Superdex 200,Pharmacia,SW)Amicon室DNA操作用酶
除非另外提及,否则DNA操作中所用的所有酶均来自New EnglandsBiolabs.Inc.,如限制性内切酶、连接酶等。方法测定IgG1阳性豚鼠的ELISA步骤
用以1∶8000稀释度稀释于碳酸盐缓冲液的兔抗PD498包被ELISA微量滴定板,4℃放置过夜。第二天这些板用2% BSA封闭1小时,再用PBS吐温20洗3次。
将1μg/ml的PD498加入板中,保温1小时,然后用PBS吐温20洗3次。
所有的豚鼠血清样品及对照都以2μl血清加98μl PBS加到ELISA平板上,保温1小时,再用PBS吐温20洗3次。
然后将山羊抗豚鼠IgG1(1∶4000稀释于PBS缓冲液(NordicImmunology 44-682))加到平板上,保温1小时,用PBS吐温20洗涤。
将碱性磷酸酶标记的兔抗山羊抗体(Sigma A4187)1∶8000稀释度样品加入滴定板上并保温1小时,在PBS吐温20中洗两次,再用二乙醇胺缓冲液洗1次。
用对硝基苯基磷酸酯在37℃保温30分钟、或直至合适的颜色显现,从而对标记的碱性磷酸酶进行显色。
用终止液(含EDTA的K2HPO4/HaH3缓冲液,pH10)终止反应,并于ELISA读数仪上读取OD 405/650数值。
双盲法被用于所有的ELISA滴定板上。
阳性和阴性血清值计算成平均盲值加两倍标准误差。这样精确度为95%。分子量的测定
用标准方法在4-20%的梯度SDS聚丙烯酰胺凝胶(Novex)上进行蛋白质的电泳分离。蛋白质用银染法检测。以来自Novex的Mark-12大范围分子量标准的迁移率为参照估测分子量。蛋白酶活性用Suc-Ala-Ala-Pro-Phe-pNa进行分析:
蛋白酶切开肽和对硝基苯胺之间的键,从而使其呈现可见的黄颜色,吸收值在405nm。
缓冲液:如Britton和Robinson缓冲液pH8.3。
底物:100mg的Suc-AAPF-pNa溶于1ml二甲亚砜(DMSO)中。用Britton和Robinson缓冲液将其中的100μl稀释至10ml。
将底物与蛋白酶溶液混和,并检测其在405nm处的吸收值,作为时间和ABS405nm/分钟的一个函数。要求控制温度(约20-50℃,具体取决于所用的蛋白酶)。这是对样品中蛋白酶活性的一个估计。蛋白水解活性
在本发明的上下文中,蛋白水解活性用Kilo Novo蛋白酶单位(KNPU)表示。相对于酶标准(SAVINASE)测定活性,依据蛋白水解酶在标准条件(即50℃,pH8.3,9分钟的反应时间,3分钟的检测时间)对二甲基酪蛋白(DMC)溶液的消化程度进行测定。如果索要,可从丹麦的Novo NordiskA/S得到一有关的折迭式印刷品AF 220/1,此印刷品特此收录于文献中。
一个GU是一个甘氨酸单位,定义为标准条件下,以N-乙酰酪蛋白为底物,40℃保温15分钟后,产生的NH2(氨基)基团量相当于1mmole甘氨酸的蛋白酶活性。
也可用PNA检测方法,根据与可溶性底物琥珀酰-丙氨酰-丙氨酰-脯氨酰-苯丙氨酰-对硝基苯酚的反应估测酶活性,此法可见于《美国石油化学协会杂志》,Rothgeb,T.M.,Goodlander,B.D.,Garrison,P.H.,和Smith,L.A.,(1988)。PD498变体的发酵
30℃下,于旋转式摇床上(300rpm),在含有100ml BPX培养液的500ml带挡板锥形瓶中摇5天,对枯草芽孢杆菌中的PD498变体进行发酵。为了得到例如2升培养液,可用20个锥形瓶同时进行发酵。培养基:BPX:组成(每升)
马铃薯淀粉 100克
磨碎大麦 50克
大豆粉 20克
Na2HPO4·12H2O 9克
Pluronic 0.1克
酪蛋白酸钠 10克
培养基中的淀粉用α-淀粉酶液化,将此培养基在120℃加热45分钟进行灭菌。灭菌后,通过加入碳酸氢钠至0.1M而将培养基的pH调至9。PD498变体的提纯
将近1.6升的PD498变体发酵液用1升离心杯于5000rpm离心35分钟。上清液用10%的乙酸将pH调至7.0,并用Seitz Supra S100滤板进行过滤。用配备了Amicon S1Y100 UF柱体的Amicon CH2A UF装置将滤液浓缩至约400ml。离心和过滤UF浓缩液后,使其在室温吸附于pH7的杆菌肽亲和柱上。用含25%2-丙醇和1M氯化钠的缓冲液室温下将PD498变体从杆菌肽柱上洗脱下来,此缓冲液还包含0.01M二甲基戊二酸、0.1M硼酸和0.002M氯化钙,pH为7。
将杆菌肽纯化步骤中有蛋白酶活性的级分合并起来,加到用含0.01M二甲基戊二酸、0.1M硼酸和0.002M氯化钙(pH6.0)的缓冲液平衡好的750ml Sephadex G25柱(直径5cm)内。
将收集自Sephadex G25柱的具蛋白水解活性的级分合并起来,加到用含0.01M二甲基戊二酸、0.1M硼酸和0.002M氯化钙(pH6.0)的缓冲液平衡好的150ml CM Sepharose CL6B阳离子交换柱(直径5厘米)上。
用1升相同缓冲液中含线性梯度0-0.5M氯化钠的溶液将蛋白酶洗脱下来。
将收集自CM Sepharose柱的含蛋白酶的级分合并在一起,用2μ滤器过滤。Balb/C小鼠IgG ELISA步骤:·用碳酸盐缓冲液稀释抗原至1mg/ml。·每一孔中加100ml。·4℃包被板过夜。·将200ml封闭缓冲液加入每一孔中,室温温育1小时,以封闭非特异性吸收。·用300ml洗涤缓冲液洗平板3次。·将未知的小鼠血清稀释于稀释缓冲液中,通常稀释10倍、20倍和40倍,或者更高倍数。·将100ml加入每一孔中。·在室温保温1小时。·用洗涤缓冲液洗涤3次,以去除未结合的物质。·用稀释缓冲液将抗小鼠IgG1抗体稀释2000倍。·将100ml加入每一孔中。·室温保温1小时。·用洗涤缓冲液洗涤3次,以去除未结合的物质。·用稀释缓冲液稀释链霉亲和素1000倍。·将100ml加入每一孔中。·室温保温1小时。·用300ml洗涤缓冲液洗涤3次,以除去未结合的物质。·将OPD(0.6mg/ml)和过氧化氢(0.4ml/ml)溶解于柠檬酸盐缓冲液中。·将100ml加入每一孔中。·室温保温10分钟。·加入100ml硫酸终止反应。·滴定板置仪器上读取492nm吸收值,以620nm吸收值为参照。小鼠的免疫
用本领域已知的标准步骤将上述修饰或未修饰多肽通过皮下注射免疫Balb/c小鼠(20克)10次(每次间隔14天)。
实施例实施例1:PD498中适于添加氨基连接基团(-NH2)的合适取代
基于与Thermitase(2tec.pdb)59%的序列同一性,如上所述构造亲本PD498的三维结构模型。
PD498的序列可见于SEQ ID NO.2。PD498的残基编号为1-280。
Insight(BIOSYM)中执行的指令可见于以下的指令文件makeKzone.bcl和makeKzone2.bcl:
保守性取代:makeKzone.bcl1 Delete Subset *2 Color Molecule Atoms * Specified Specification 55,0,2553 Zone Subset LYS:lys:NZ Static monomer/residue 10Color_Subset 255,255,04 Zone Subset NTERM:1:N Static monomer/residue 10Color_Subset 255,255,05 #NOTE:editnextline ACTSITE residues according to theprotein6 Zone Subset ACTSITE:39,72,226 Static monomer/residue 8Color_Subset 255,255,07 Combine Subset ALLZONE Union LYS NTERM8 Combine Subset ALLZONE Union ALLZONE ACTSITE9 #NOTE:editnextline object name according to the protein10 Combine Subset REST Difference PD498FINALMODEL ALLZONE11 List Subset REST Atom Output_File restatom.list12 List Subset REST monomer/residue Output_File restmole.list13 Color Molecule Atoms ACTSITE Specified Specification 255,0,014 List Subset ACTSITE Atom Output_File actsiteatom.list15 List Subset ACTSITE monomer/residue Output_Fileactsitemole.list16 #17 Zone Subset REST5A REST Static Monomer/Residue 5 -Color_Subset18 Combine Subset SUB5A Difference REST5A ACTSITE19 Combine Subset SUB5B Difference SUB5A REST20 Color Molecule Atoms SUB5B Specified Specification255,255,25521 List Subset SUB5B Atom Output_File sub5batom.list22 List Subset SUB5B monomer/residue Output_File sub5bmole.list23 #Now identify sites for lys→arg substitutions and continuewith makezone2.bcl24 #Use grep command to identify ARG in restatom.list,sub5batom.list & accsiteatom.list说明:
第1-8行:ALLZONE亚组定义为所处位置距离赖氨酸上游离氨基或N-末端10以内,或者距离催化性三联体残基39、72和226 8以内的那些残基。
第10行:REST亚组定义为不包括在ALLZONE内的那些残基。
第17-20行:SUB5B亚组定义为位于REST周围一个5的壳内、除去距离催化性残基8以内的残基的那些残基。
第23-24行:REST包含Arg62和Arg169,SUB5B包含Arg51,Arg121和Arg250。ACTSITE包含Arg103,但位置103距离必需的催化性残基在8以内,因此并不相关。
彩色残基有:(255,0,255)=深红色,(255,255,0)黄色,(255,0,0)红色,和(255,255,255)=白色。
已鉴定出R51K,R62K,R121K,R169K和R250K这些取代是亲本PD498中适于诱变的一些合适位点。在下面的第2部分将对这些残基进行取代,并进一步分析:
非保守性取代:makeKzone2.bcl1 #sourcefile makezone2.bcl Claus von der Osten 9611282 #3 #having scanned lists (grep arg command)and identifiedsites for lys→arg substitutions4 #NOTE:editnextline object name according to protein5 Copy Object -To_Clipboard -Displace PD498FINALMODELnewmodel6 Biopolymer7 #NOTE:editnextline object name according to protein8 Blank Object On PD498FINALMODEL9 #NOTE:editnextlines with lys→arg positions10 Replace Residue newmodel:51 lys L11 Replace Residue newmodel:62 lys L12 Replace Residue newmodel:121 lys L13 Replace Residue newmodel:169 lys L14 Replace Residue newmodel:250 lys L15 #16 #Now repeat analysis done prior to arg→lys,now includingintroduced lysines17 Color Molecule Atoms newmodel Specified Specification255,0,25518 Zone Subset LYSx newmodel:lys:NZ Static monomer/residue 10Color_Subset 255,255,019 Zone Subset NTERMx newmodel:l:N Static monomer/residue 10Color_Subset 255,255,020 #NOTE:editnextline ACTSITEx residues according to theprotein21 Zone Subset ACTSITEx newmodel:39,72,226 Staticmonomer/residue 8 Color_Subset 255,255,022 Combine Subset ALLZONEx Union LYSx NTERMx23 Combine Subset ALLZONEx Union ALLZONEx ACTSITEx24 Combine Subset RESTx Difference newmodel ALLZONEx25 List Subset RESTx Atom Output_File restxatom.list26 List Subset RESTx monomer/residue Output_Filerestxmole.list27 #28 Color Molecule Atoms ACTSITEx Specified Specification255,0,029 List Subset ACTSITEx Atom Output File actsitexatom.list30 List Subset ACTSITEx monomer/residue Output_Fileactsitexmole.list31 #32 #read restxatom.list or restxmole.list to identify sitesfor (not_arg)→lys subst.if needed说明:
第1-15行:REST和SUB5B亚组中溶剂暴露型精氨酸被赖氨酸所取代。精氨酸取代后,重新计算溶剂可及性。
第16-23行:ALLZONEx亚组定义为所处位置距离赖氨酸(取代后)上游离氨基或N-末端10以内、或者距离催化性三联体残基39、72和226 8以内的那些残基。
第24-26行:RESTx亚组定义为不包括在ALLZONEx内的那些残基,即仍有可能构成表位的残基。在RESTx亚组内,下述残基暴露程度>5%(见下表):6-7,9-12,43-45,65,87-88,209,211,216-221,262。
建议在亲本PD498内进行下述突变:P6K,Y7K,S9K,A10K,Y11K,Q12K,D43K,Y44K,N45K,N65K,G87K,I88K,N209K,A211K,N216K,N217K,G218K,Y219K,S220K,Y221K,G262K。
实施例1中的有关数据:
PD498MODEL的溶剂可及性数据:#PD498MODEL Fri Nov 29 10:24:48 MET 1996#残基 面积TRP_1 136.275711SER_2 88.188095PRO_3 15.458788ASN_4 95.322319ASP_5 4.903404PRO_6 68.096909TYR_7 93.333252TYR_8 31.791576SER_9 95.983139ALA_10 77.983536TYR_11 150.704727GLN_12 26.983349TYR_13 44.328232GLY_14 3.200084PRO_15 2.149547GLN_16 61.385445ASN_17 37.776707THR_18 1.237873SER_19 41.031750THR_20 4.321402PRO_21 16.658991ALA_22 42.107288ALA_23 0.000000TRP_24 3.713619ASP_25 82.645493VAL_26 74.397812THR_27 14.950654ARG_28 110.606209GLY_29 0.242063SER_30 57.225292SER_31 86.986198THR_32 1.928865GLN_33 42.008949THR_34 0.502189VAL_35 0.268693ALA_36 0.000000VAL_37 5.255383LEU_38 1.550332ASP_39 3.585718SER_40 2.475746GLY_41 4.329043VAL_42 1.704864ASP_43 25.889742TYR_44 89.194855ASN_45 109.981819HIS_46 0.268693PRO_47 66.580925ASP_48 0.000000LEU_49 0.770882ALA_50 49.618046ARG_51 218.751709LYS_52 18.808538VAL_53 39.937984ILE_54 98.478104LYS_55 103.612228GLY_56 17.199390TYR_57 67.719147ASP_58 0.000000PHE_59 40.291119ILE_60 50.151962ASP_61 70.078888ARG_62 166.777557ASP_63 35.892376ASN_64 120.641953ASN_65 64.982895PRO_66 6.986028MET_67 58.504269ASP_68 28.668840LEU_69 104.467468ASN_70 78.460953GLY_71 5.615932HIS_72 43.158905GLY_73 0.268693THR_74 0.000000HIS_75 0.484127VAL_76 1.880854ALA_77 0.000000GLY_78 0.933982THR_79 9.589676VAL_80 0.000000ALA_81 0.000000ALA_82 0.000000ASP_83 46.244987THR_84 27.783333ASN_85 75.924225ASN_86 44.813908GLY_87 50.453152ILE_88 74.428070GLY_89 4.115077VAL_90 6.717335ALA_91 2.872341GLY_92 0.233495MET_93 5.876057ALA_94 0.000000PRO_95 17.682203ASP_96 83.431740THR_97 1.506567LYS_98 72.674973ILE_99 4.251006LEU_100 6.717335ALA_101 0.806080VAL_102 1.426676ARG_103 2.662697VAL_104 2.171855LEU_105 18.808538ASP_106 52.167435ALA_107 52.905663ASN_108 115.871315GLY_109 30.943356SER_110 57.933651GLY_111 50.705326SER_112 56.383320LEU_113 71.312195ASP_114 110.410919SER_115 13.910152ILE_116 22.570246ALA_117 5.642561SER_118 29.313131GLY_119 0.000000ILE_120 1.343467ARG_121 118.391129TYR_122 44.203033ALA_123 0.000000ALA_124 7.974043ASP_125 83.851639GLN_126 64.311974GLY_127 36.812618ALA_128 4.705107LYS_129 90.886139VAL_130 1.039576LEU_131 2.149547ASN_132 4.315227LEU_133 1.880854SER_134 3.563334LEU_135 26.371397GLY_136 59.151070CYS_137 63.333755GLU_138 111.553314CYS_139 83.591461ASN_140 80.757843SER_141 25.899158THR_142 99.889725THR_143 73.323814LEU_144 5.589301LYS_145 94.708755SER_146 72.636993ALA_147 9.235920VAL_148 1.612160ASP_149 57.431465TYR_150 106.352493ALA_151 0.268693TRP_152 43.133667ASN_153 112.864975LYS_154 110.009468GLY_155 33.352180ALA_156 3.493014VAL_157 1.048144VAL_158 2.043953VAL_159 0.000000ALA_160 0.537387ALA_161 10.872165ALA_162 7.823834GLY_163 12.064573ASN_164 81.183388ASP_165 64.495300ASN_166 83.457443VAL_167 68.516815SER_168 78.799652ARG_169 116.937134THR_170 57.275074PHE_171 51.416462GLN_172 18.934589PRO_173 1.880854ALA_174 6.522357SER_175 26.184139TYR_176 21.425076PRO_177 85.613541ASN_178 34.700817ALA_179 0.268693ILE_180 1.074774ALA_181 3.761708VAL_182 0.000000GLY_183 2.149547ALA_184 0.951118ILE_185 0.806080ASP_186 30.022263SER_187 72.518509ASN_188 117.128021ASP_189 47.601345ARG_190 150.050873LYS_191 64.822807ALA_192 2.686934SER_193 96.223808PHE_194 51.482613SER_195 1.400973ASN_196 4.148808TYR_197 80.937309GLY_198 10.747736THR_199 93.221252TRP_200 169.943604VAL_201 15.280325ASP_202 12.141763VAL_203 0.268693THR_204 3.409728ALA_205 0.000000PRO_206 0.000000GLY_207 0.000000VAL_208 37.137192ASN_209 78.286270ILE_210 9.404268ALA_211 25.938599SER_212 5.037172THR_213 0.000000VAL_214 22.301552PRO_215 45.251030ASN_216 131.014160ASN_217 88.383461GLY_218 21.226780TYR_219 88.907570SER_220 39.966541TYR_221 166.037018MET_222 50.951096SER_223 54.435001GLY_224 1.880854THR_225 1.634468SER_226 17.432346MET_227 7.233279ALA_228 0.000000SER_229 0.000000PRO_230 0.268693HIS_231 2.680759VAL_232 0.000000ALA_233 0.000000GLY_234 1.074774LEU_235 11.500556ALA_236 0.000000ALA_237 0.000000LEU_238 1.612160LEU_239 0.000000ALA_240 10.648088SER_241 39.138004GLN_242 71.056175GLY_243 66.487144LYS_244 43.256012ASN_245 80.728127ASN_246 34.859673VAL_247 84.145645GLN_248 51.819775ILE_249 8.598188ARG_250 35.055809GLN_251 71.928093ALA_252 0.000000ILE_253 4.845899GLU_254 13.344438GLN_255 81.705254THR_256 9.836061ALA_257 2.810513ASP_258 44.656136LYS_259 113.071686ILE_260 32.089527SER_261 91.590103GLY_262 26.450439THR_263 38.308762GLY_264 46.870056THR_265 88.551804ASN_266 34.698349PHE_267 7.756911LYS_268 103.212852TYR_269 37.638382GLY_270 0.000000LYS_271 11.376978ILE_272 2.885231ASN_273 19.195255SER_274 2.651736ASN_275 38.177547LYS_276 84.549576ALA_277 1.074774VAL_278 4.775503ARG_279 162.693054TYR_280 96.572929CA_281 0.000000CA_282 0.000000CA_283 8.803203Subset REST: restmole.listSubset REST:PD498FINALMODEL:6-7,9-12,43-46,61-63,65,87-89,111-114,117-118,131,PD498FINALMODEL:137-139,158-159,169-171,173-174,180-181,209,211,PD498FINALMODEL:216-221,232-233,262,E282Hrestatom.listSubset REST:
PD498FINALMODEL:PRO 6:N,CA,CD,C,O,CB,CG
PD498FINALMODEL:TYR 7:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH
PD498FINALMODEL:SER 9:N,CA,C,O,CB,OG
PD498FINALMODEL:ALA 10:N,CA,C,O,CB
PD498FINALMODEL:TYR 11:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH
PD498FINALMODEL:GLN 12:N,CA,C,O,CB,CG,CD,OE1,NE2
PD498FINALMODEL:ASP 43:N,CA,C,O,CB,CG,OD1,OD2
PD498FINALMODEL:TYR
44:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH
PD498FINALMODEL:ASN 45:N,CA,C,O,CB,CG,OD1,ND2
PD498FINALMODEL:HIS
46:N,CA,C,O,CB,CG,ND1,CD2,CE1,NE2
PD498FINALMODEL:ASP 61:N,CA,C,O,CB,CG,OD1,OD2
PD498FINALMODEL:ARG
62:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2
PD498FINALMODEL:ASP 63:N,CA,C,O,CB,CG,OD1,OD2
PD498FINALMODEL:ASN 65:N,CA,C,O,CB,CG,OD1,ND2
PD498FINALMODEL:GLY 87:N,CA,C,O
PD498FINALMODEL:ILE 88:N,CA,C,O,CB,CG1,CG2,CD1
PD498FINALMODEL:GLY 89:N,CA,C,O
PD498FINALMODEL:GLY 111:N,CA,C,O
PD498FINALMODEL:SER 112:N,CA,C,O,CB,OG
PD498FINALMODEL:LEU 113:N,CA,C,O,CB,CG,CD1,CD2
PD498FINALMODEL:ASP 114:N,CA,C,O,CB,CG,OD1,OD2
PD498FINALMODEL:ALA 117:N,CA,C,O,CB
PD498FINALMODEL:SER 118:N,CA,C,O,CB,OG
PD498FINALMODEL:LEU 131:N,CA,C,O,CB,CG,CD1,CD2
PD498FINALMODEL:CYS 137:N,CA,C,O,CB,SG
PD498FINALMODEL:GLU
138:N,CA,C,O,CB,CG,CD,OE1,OE2
PD498FINALMODEL:CYS 139:N,CA,C,O,CB,SG
PD498FINALMODEL:VAL 158:N,CA,C,O,CB,CG1,CG2
PD498FINALMODEL:VAL 159:N,CA,C,O,CB,CG1,CG2
PD498FINALMODEL:ARG
169:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2
PD498FINALMODEL:THR 170:N,CA,C,O,CB,OG1,CG2
PD498FINALMODEL:PHE
171:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ
PD498FINALMODEL:PRO 173:N,CA,CD,C,O,CB,CG
PD498FINALMODEL:ALA 174:N,CA,C,O,CB
PD498FINALMODEL:ILE 180:N,CA,C,O,CB,CG1,CG2,CD1
PD498FINALMODEL:ALA 181:N,CA,C,O,CB
PD498FINALMODEL:ASN 209:N,CA,C,O,CB,CG,OD1,ND2
PD498FINALMODEL:ALA 211:N,CA,C,O,CB
PD498FINALMODEL:ASN 216:N,CA,C,O,CB,CG,OD1,ND2
PD498FINALMODEL:ASN 217:N,CA,C,O,CB,CG,OD1,ND2
PD498FINALMODEL:GLY 218:N,CA,C,O
PD498FINALMODEL:TYR
219:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH
PD498FINALMODEL:SER 220:N,CA,C,O,CB,OG
PD498FINALMODEL:TYR
221:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH
PD498FINALMODEL:VAL 232:N,CA,C,O,CB,CG1,CG2
PD498FINALMODEL:ALA 233:N,CA,C,O,CB
PD498FINALMODEL:GLY 262:N,CA,C,O
PD498FINALMODEL:CA E282H:CASubset SUB5B:sub5bmole.listSubset SUB5B:
PD498FINALMODEL:4-5,8,13-16,34-35,47-51,53,64,83,85-86,90-91,120-124,
PD498FINALMODEL:128-130,140-141,143-144,147-148,151-152,156-157,
PD498FINALMODEL:165,167-168,172,175-176,178-179,196,200-205,208,
PD498FINALMODEL:234-237,250,253-254,260-261,263-267,272,E281H,
PD498FINALMODEL:E283Hsub5batom.listSubset SUB5B:
PD498FINALMODEL:ASN 4:N,CA,C,O,CB,CG,OD1,ND2
PD498FINALMODEL:ASP 5:N,CA,C,O,CB,CG,OD1,OD2
PD498FINALMODEL:TYR
8:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH
PD498FINALMODEL:TYR
13:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH
PD498FINALMODEL:GLY 14:N,CA,C,O
PD498FINALMODEL:PRO 15:N,CA,CD,C,O,CB,CG
PD498FINALMODEL:GLN 16:N,CA,C,O,CB,CG,CD,OE1,NE2
PD498FINALMODEL:THR 34:N,CA,C,O,CB,OG1,CG2
PD498FINALMODEL:VAL 35:N,CA,C,O,CB,CG1,CG2
PD498FINALMODEL:PRO 47:N,CA,CD,C,O,CB,CG
PD498FINALMODEL:ASP 48:N,CA, C,O,CB,CG,OD1,OD2
PD498FINALMODEL:LEU 49:N,CA,C,O,CB,CG,CD1,CD2
PD498FINALMODEL:ALA 50:N,CA,C,O,CB
PD498FINALMODEL:ARG
51:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2
PD498FINALMODEL:VAL 53:N,CA,C,O,CB,CG1,CG2
PD498FINALMODEL:ASN 64:N,CA,C,O,CB,CG,OD1,ND2
PD498FINALMODEL:ASP 83:N,CA,C,O,CB,CG,OD1,OD2
PD498FINALMODEL:ASN 85:N,CA,C,O,CB,CG,OD1,ND2
PD498FINALMODEL:ASN 86:N,CA,C,O,CB,CG,OD1,ND2
PD498FINALMODEL:VAL 90:N,CA,C,O,CB,CG1,CG2
PD498FINALMODEL:ALA 91:N,CA,C,O,CB
PD498FINALMODEL:ILE 120:N,CA,C,O,CB,CG1,CG2,CD1
PD498FINALMODEL:ARG
121:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2
PD498FINALMODEL:TYR
122:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH
PD498FINALMODEL:ALA 123:N,CA,C,O,CB
PD498FINALMODEL:ALA 124:N,CA,C,O,CB
PD498FINALMODEL:ALA 128:N,CA,C,O,CB
PD498FINALMODEL:LYS 129:N,CA,C,O,CB,CG,CD,CE,NZ
PD498FINALMODEL:VAL 130:N,CA,C,O,CB,CG1,CG2
PD498FINALMODEL:ASN 140:N,CA,C,O,CB,CG,OD1,ND2
PD498FINALMODEL:SER 141:N,CA,C,O,CB,OG
PD498FINALMODEL:THR 143:N,CA,C,O,CB,OG1,CG2
PD498FINALMODEL:LEU 144:N,CA,C,O,CB,CG,CD1,CD2
PD498FINALMODEL:ALA 147:N,CA,C,O,CB
PD498FINALMODEL:VAL 148:N,CA,C,O,CB,CG1,CG2
PD498FINALMODEL:ALA 151:N,CA,C,O,CB
PD498FINALMODEL:TRP
52:N,CA,C,O,CB,CG,CD1,CD2,NE1,CE2,CE3,
CZ2,CZ3,CH2
PD498FINALMODEL:ALA 156:N,CA,C,O,CB
PD498FINALMODEL:VAL 157:N,CA,C,O,CB,CG1,CG2
PD498FINALMODEL:ASP 165:N,CA,C,O,CB,CG,OD1,OD2
PD498FINALMODEL:VAL 167:N,CA,C,O,CB,CG1,CG2
PD498FINALMODEL:SER 168:N,CA,C,O,CB,OG
PD498FINALMODEL:GLN
172:N,CA,C,O,CB,CG,CD,OE1,NE2
PD498FINALMODEL:SER 175:N,CA,C,O,CB,OG
PD498FINALMODEL:TYR
176:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH
PD498FINALMODEL:ASN 178:N,CA,C,O,CB,CG,OD1,ND2
PD498FINALMODEL:ALA 179:N,CA,C,O,CB
PD498FINALMODEL:ASN 196:N,CA,C,O,CB,CG,OD1,ND2
PD498FINALMODEL:TRP
200:N,CA,C,O,CB,CG,CD1,CD2,NE1,CE2,CE3,
CZ2,CZ3,CH2
PD498FINALMODEL:VAL 201:N,CA,C,O,CB,CG1,CG2
PD498FINALMODEL:ASP 202:N,CA,C,O,CB,CG,OD1,OD2
PD498FINALMODEL:VAL 203:N,CA,C,O,CB,CG1,CG2
PD498FINALMODEL:THR 204:N,CA,C,O,CB,OG1,CG2
PD498FINALMODEL:ALA 205:N,CA,C,O,CB
PD498FINALMODEL:VAL 208:N,CA,C,O,CB,CG1,CG2
PD498FINALMODEL:GLY 234:N,CA,C,O
PD498FINALMODEL:LEU 235:N,CA,C,O,CB,CG,CD1,CD2
PD498FINALMODEL:ALA 236:N,CA,C,O,CB
PD498FINALMODEL:ALA 237:N,CA,C,O,CB
PD498FINALMODEL:ARG
250:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2
PD498FINALMODEL:ILE 253:N,CA,C,O,CB,CG1,CG2,CD1
PD498FINALMODEL:GLU
254:N,CA,C,O,CB,CG,CD,OE1,OE2
PD498FINALMODEL:ILE 260:N,CA,C,O,CB,CG1,CG2,CD1
PD498FINALMODEL:SER 261:N,CA,C,O,CB,OG
PD498FINALMODEL:THR 263:N,CA,C,O,CB,OG1,CG2
PD498FINALMODEL:GLY 264:N,CA,C,O
PD498FINALMODEL:THR 265:N,CA,C,O,CB,OG1,CG2
PD498FINALMODEL:ASN 266:N,CA,C,O,CB,CG,OD1,ND2
PD498FINALMODEL:PHE
267:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ
PD498FINALMODEL:ILE 272:N,CA,C,O,CB,CG1,CG2,CD1
PD498FINALMODEL:CA E281H:CA
PD498FINALMODEL:CA E283H:NASubset ACTSITE:
actsitemole.listSubset ACTSITE:
PD498FINALMODEL:36-42,57-60,66-80,100-110,115-116,119,132-136,160-164,
PD498FINALMODEL:182-184,194,206-207,210,212-215,222-231actsiteatom.listSubset ACTSITE:
PD498FINALMODEL:ALA 36:N,CA,C,O,CB
PD498FINALMODEL:VAL 37:N,CA,C,O,CB,CG1,CG2
PD498FINALMODEL:LEU 38:N,CA,C,O,CB,CG,CD1,CD2
PD498FINALMODEL:ASP 39:N,CA,C,O,CB,CG,OD1,OD2
PD498FINALMODEL:SER 40:N,CA,C,O,CB,OG
PD498FINALMODEL:GLY 41:N,CA,C,O
PD498FINALMODEL:VAL 42:N,CA,C,O,CB,CG1,CG2
PD498FINALMODEL:TYR
57:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH
PD498FINALMODEL:ASP 58:N,CA,C,O,CB,CG,OD1,OD2
PD498FINALMODEL:PHE
59:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ
PD498FINALMODEL:ILE 60:N,CA,C,O,CB,CG1,CG2,CD1
PD498FINALMODEL:PRO 66:N,CA,CD,C,O,CB,CG
PD498FINALMODEL:MET 67:N,CA,C,O,CB,CG,SD,CE
PD498FINALMODEL:ASP 68:N,CA,C,O,CB,CG,OD1,OD2
PD498FINALMODEL:LEU 69:N,CA,C,O,CB,CG,CD1,CD2
PD498FINALMODEL:ASN 70:N,CA,C,O,CB,CG,OD1,ND2
PD498FINALMODEL:GLY 71:N,CA,C,O
PD498FINALMODEL:HIS
72:N,CA,C,O,CB,CG,ND1,CD2,CE1,NE2
PD498FINALMODEL:GLY 73:N,CA,C,O
PD498FINALMODEL:THR 74:N,CA,C,O,CB,OG1,CG2
PD498FINALMODEL:HIS
75:N,CA,C,O,CB,CG,ND1,CD2,CE1,NE2
PD498FINALMODEL:VAL 76:N,CA,C,O,CB,CG1,CG2
PD498FINALMODEL:ALA 77:N,CA,C,O,CB
PD498FINALMODEL:GLY 78:N,CA,C,O
PD498FINALMODEL:THR 79:N,CA,C,O,CB,OG1,CG2
PD498FINALMODEL:VAL 80:N,CA,C,O,CB,CG1,CG2
PD498FINALMODEL:LEU 100:N,CA,C,O,CB,CG,CD1,CD2
PD498FINALMODEL:ALA 101:N,CA,C,O,CB
PD498FINALMODEL:VAL 102:N,CA,C,O,CB,CG1,CG2
PD498FINALMODEL:ARG
103:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2
PD498FINALMODEL:VAL 104:N,CA,C,O,CB,CG1,CG2
PD498FINALMODEL:LEU 105:N,CA,C,O,CB,CG,CD1,CD2
PD498FINALMODEL:ASP 106:N,CA,C,O,CB,CG,OD1,OD2
PD498FINALMODEL:ALA 107:N,CA,C,O,CB
PD498FINALMODEL:ASN 108:N,CA,C,O,CB,CG,OD1,ND2
PD498FINALMODEL:GLY 109:N,CA,C,O
PD498FINALMODEL:SER 110:N,CA,C,O,CB,OG
PD498FINALMODEL:SER 115:N,CA,C,O,CB,OG
PD498FINALMODEL:ILE 116:N,CA,C,O,CB,CG1,CG2,CD1
PD498FINALMODEL:GLY 119:N,CA,C,O
PD498FINALMODEL:ASN 132:N,CA,C,O,CB,CG,OD1,ND2
PD498FINALMODEL:LEU 133:N,CA,C,O,CB,CG,CD1,CD2
PD498FINALMODEL:SER 134:N,CA,C,O,CB,OG
PD498FINALMODEL:LEU 135:N,CA,C,O,CB,CG,CD1,CD2
PD498FINALMODEL:GLY 136:N,CA,C,O
PD498FINALMODEL:ALA 160:N,CA,C,O,CB
PD498FINALMODEL:ALA 161:N,CA,C,O,CB
PD498FINALMODEL:ALA 162:N,CA,C,O,CB
PD498FINALMODEL:GLY 163:N,CA,C,O
PD498FINALMODEL:ASN 164:N,CA,C,O,CB,CG,OD1,ND2
PD498FINALMODEL:VAL 182:N,CA,C,O,CB,CG1,CG2
PD498FINALMODEL:GLY 183:N,CA,C,O
PD498FINALMODEL:ALA 184:N,CA,C,O,CB
PD498FINALMODEL:PHE
194:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ
PD498FINALMODEL:PRO 206:N,CA,CD,C,O,CB,CG
PD498FINALMODEL:GLY 207:N,CA,C,O
PD498FINALMODEL:ILE 210:N,CA,C,O,CB,CG1,CG2,CD1
PD498FINALMODEL:SER 212:N,CA,C,O,CB,OG
PD498FINALMODEL:THR 213:N,CA,C,O,CB,OG1,CG2
PD498FINALMODEL:VAL 214:N,CA,C,O,CB,CG1,CG2
PD498FINALMODEL:PRO 215:N,CA,CD,C,O,CB,CG
PD498FINALMODEL:MET 222:N,CA,C,O,CB,CG,SD,CE
PD498FINALMODEL:SER 223:N,CA,C,O,CB,OG
PD498FINALMODEL:GLY 224:N,CA,C,O
PD498FINALMODEL:THR 225:N,CA,C,O,CB,OG1,CG2
PD498FINALMODEL:SER 226:N,CA,C,O,CB,OG
PD498FINALMODEL:MET 227:N,CA,C,O,CB,CG,SD,CE
PD498FINALMODEL:ALA 228:N,CA,C,O,CB
PD498FINALMODEL:SER 229:N,CA,C,O,CB,OG
PD498FINALMODEL:PRO 230:N,CA,CD,C,O,CB,CG
PD498FINALMODEL:HIS
231:N,CA,C,O,CB,CG,ND1,CD2,CE1,NE2Subset RESTx:restxmole.listSubset RESTX:
NEWMODEL:6-7,9-12,43-46,65,87-89,131,173,209,211,216-221,232-233,
NEWMODEL:262,E282Hrestxatom.listSubset RESTX:
NEWMODEL:PRO 6:N,CA,CD,C,O,CB,CG
NEWMODEL:TYR7:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH
NEWMODEL:SER 9:N,CA,C,O,CB,OG
NEWMODEL:ALA 10:N,CA,C,O,CB
NEWMODEL:TYR11:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH
NEWMODEL:GLN 12:N,CA,C,O,CB,CG,CD,OE1,NE2
NEWMODEL:ASP 43:N,CA,C,O,CB,CG,OD1,OD2
NEWMODEL:TYR44:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH
NEWMODEL:ASN 45:N,CA,C,O,CB,CG,OD1,ND2
NEWMODEL:HIS 46:N,CA,C,O,CB,CG,ND1,CD2,CE1,NE2
NEWMODEL:ASN 65:N,CA,C,O,CB,CG,OD1,ND2
NEWMODEL:GLY 87:N,CA,C,O
NEWMODEL:ILE 88:N,CA,C,O,CB,CG1,CG2,CD1
NEWMODEL:GLY 89:N,CA,C,O
NEWMODEL:LEU 131:N,CA,C,O,CB,CG,CD1,CD2
NEWMODEL:PRO 173:N,CA,CD,C,O,CB,CG
NEWMODEL:ASN 209:N,CA,C,O,CB,CG,OD1,ND2
NEWMODEL:ALA 211:N,CA,C,O,CB
NEWMODEL:ASN 216:N,CA,C,O,CB,CG,OD1,ND2
NEWMODEL:ASN 217:N,CA,C,O,CB,CG,OD1,ND2
NEWMODEL:GLY 218:N,CA,C,O
NEWMODEL:TYR219:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH
NEWMODEL:SER 220:N,CA,C,O,CB,OG
NEWMODEL:TYR221:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH
NEWMODEL:VAL 232:N,CA,C,O,CB,CG1,CG2
NEWMODEL:ALA 233:N,CA,C,O,CB
NEWMODEL:GLY 262:N,CA,C,O
NEWMODEL:CA E282H:CA实施例2:Savinase中适于添加氨基连接基团(-NH2)的合适取代
利用Savinase的已知X-射线结构,找出在何处可以添加合适的氨基连接基团(Betzel等,1992,分子生物学杂志,223,427-445)。
在Brookhaven数据库中,可以1svn.pbd的文件名得到Savinase的3D结构。
Savinase的序列示于SEQ ID NO:3中。所用的序列编号与枯草杆菌蛋白酶BPN’中的一致,相对于BPN’,Savinase在36、56、158-159和163-164这些位置处存在缺失。活性位点残基(功能性位点)是D32、H64和S221。
Insight(BIOSYM)中运行的各个指令示于下述指令文件makeKzone.bcl和makeKzone2.bcl中:
保守性取代:makeKzone.bclDelete Subset *Color Molecule Atoms * Specified Specification 255,0,255Zone Subset LYS:lys:NZ Static monomer/residue 10 Color_Subset255,255,0Zone Subset NTERM:e1:N Static monomer/residue 10 Color_Subset255,255,0#NOTE:editnextline ACTSITE residues according to the proteinZone Subset ACTSITE:e32,e64,e221 Static monomer/residue 8Color_Subset 255,255,0Combine Subset ALLZONE Union LYS NTERMCombine Subset ALLZONE Union ALLZONE ACTSITE#NOTE:editnextline object name according to the proteinCombine Subset REST Difference SAVI8 ALLZONEList Subset REST Atom Output File_restatom.listList Subset REST monomer/residue Output_File restmole.listColor Molecule Atoms ACTSITE Specified Specification 255,0,0List Subset ACTSITE Atom Output_File actsiteatom.listList Subset ACTSITE monomer/residue Output_Fileactsitemole.list#Zone Subset REST5A REST Static Monomer/Residue 5 -Color SubsetCombine Subset SUB5A Difference REST5A ACTSITECombine Subset SUB5B Difference SUB5A RESTColor Molecule Atoms SUB5B Specified Specification 255,255,255List Subset SUB5B Atom Output_File sub5batom.listList Subset SUB5B monomer/residue Output_File sub5bmole.list#Now identify sites for lys→arg substitutions and continuewith makezone2.bcl#Use grep command to identify ARG in restatom.list,sub5batom.list & accsiteatom.list说明:
在Savinase的情形下,REST包含精氨酸Arg10,Arg170和Arg186,SUB5B包含Arg19,Arg45,Arg145和Arg247。
这些残基均是溶剂暴露型的。已鉴定出Savinase内R10K,R19K,R45K,R145K,R170K,R186K和R247K这些取代是适于进行本发明范围内诱变的一些位点。在下述第2部分将对这些残基进行取代,并进一步分析。ACTSITE亚组包含Lys94。
K94R这个取代是能除去作为活性位点附近连接基团的赖氨酸的一个突变。
非保守性取代:makeKzone2.bcl#sourcefile makezone2.bcl Claus von der Osten 961128##having scanned lists(grep arg command) and identified sitesfor lys→arg substitutions#NOTE:editnextline object name according to proteinCopy Object -To_Clipboard -Displace SAVI8 newmodelBiopolymer#NOTE:editnextline object name according to proteinBlank Object On SAVI8#NOTE:editnextlines with lys→arg positionsReplace Residue newmodel:e10 lys LReplace Residue newmodel:e170 lys LReplace Residue newmodel:e186 lys LReplace Residue newmodel:e19 lys LReplace Residue newmodel:e45 lys LReplace Residue newmodel:e145 lys LReplace Residue newmodel:e241 lys L##Now repeat analysis done prior to arg→lys,now includingintroduced lysinesColor Molecule Atoms newmodel Specified Specification 255,0,255Zone Subset LYSx newmodel:lys:NZ Static monomer/residue 10Color_Subset 255,255,0Zone Subset NTERMx newmodel:e1:N Static monomer/residue 10Color_Subset 255,255,0#NOTE:editnextline ACTSITEx residues according to the proteinZone Subset ACTSITEx newmodel:e32,e64,e221 Staticmonomer/residue 8 Color_Subset 255,255,0Combine Subset ALLZONEx Union LYSx NTERMxCombine Subset ALLZONEx Union ALLZONEx ACTSITExCombine Subset RESTx Difference newmodel ALLZONExList Subset RESTx Atom Output_File restxatom.listList Subset RESTx monomer/residue Output_File restxmole.list#Color Molecule Atoms ACTSITEx Specified Specification 255,0,0List Subset ACTSITEx Atom Output_File actsitexatom.listList Subset ACTSITEx monomer/residue Output_Fileactsitexmole.list##read restxatom.list or restxmole.list to identify sites for(not_arg)→lys subst. if needed说明:
在RESTx亚组内,下述残基暴露程度>5%(见下表):5,14,22,38-40,42,75-76,82,86,103-105,108,133-135,137,140,173,204,206,211-213,215-216,269。建议在Savinase中进行下述突变: P5K,P14K,T22K,T38K,H39K,P40K,L42K,L75K,N76K,L82K,P86K,S103K,V104K,S105K,A108K,A133K,T134K,L135K,Q137K,N140K,N173K,N204K,Q206K,G211K,S212K,T213K,A215K,S216K,N269K。
实施例2的有关数据:
SAVINASE中的溶剂可及性数据:# SAVI8NOH2O Fri Nov 29 13:32:07 MET 1996#残基 面积ALA_1 118.362808GLN_2 49.422764SER_3 61.982887VAL_4 71.620255PRO_5 21.737535TRP_6 58.718731GLY_7 4.328117ILE_8 6.664074SER_9 60.175900ARG_10 70.928963VAL_11 2.686934GLN_12 72.839996ALA_13 0.000000PRO_14 52.308453ALA_15 38.300892ALA_16 0.000000HIS_17 41.826324ASN_18 136.376602ARG_19 105.678642GLY_20 48.231510LEU_21 17.196377THR_22 36.781742GLY_23 0.000000SER_24 64.151276GLY_25 50.269905VAL_26 4.030401LYS_27 54.239555VAL_28 0.000000ALA_29 0.000000VAL_30 3.572827LEU_31 0.233495ASP_32 1.074774THR_33 1.973557GLY_34 3.638052ILE_35 8.044439SER_36 8.514903THR_37 122.598907HIS_38 18.834011PRO_39 76.570526ASP_40 0.000000LEU_41 19.684013ASN_42 88.870216ILE_43 56.117710ARG_44 110.647194GLY_45 26.935413GLY_46 35.515778ALA_47 21.495472SER_48 34.876190PHE_49 52.647541VAL_50 23.364208PRO_51 110.408752GLY_52 80.282906GLU_53 43.033707PRO_54 124.444336SER_55 60.284889THR_56 47.103241GLN_57 120.803505ASP_58 12.784743GLY_59 61.742443ASN_60 56.760231GLY_61 1.576962HIS_62 38.590118GLY_63 0.000000THR_64 0.537387HIS_65 0.968253VAL_66 1.612160ALA_67 0.000000GLY_68 2.801945THR_69 9.074596ILE_70 0.000000ALA_71 4.577205ALA_72 0.000000LEU_73 47.290039ASN_74 102.187248ASN_75 60.210400SER_76 84.614494ILE_77 66.098572GLY_78 17.979534VAL_79 5.642561LEU_80 13.025185GLY_81 0.000000VAL_82 0.268693ALA_83 0.000000PRO_84 18.193810SER_85 56.839039ALA_86 13.075745GLU_87 37.011765LEU_88 2.149547TYR_89 30.633518ALA_90 1.343467VAL_91 0.779450LYS_92 5.862781VAL_93 0.466991LEU_94 10.747736GLY_95 8.707102ALA_96 41.414677SER_97 96.066040GLY_98 33.374485SER_99 67.664116GLY_100 35.571117SER_101 54.096992VAL_102 52.695324SER_103 62.929684SER_104 8.683097ILE_105 15.852910ALA_106 14.509443GLN_107 94.463066GLY_108 0.000000LEU_109 0.537387GLU_110 63.227707TRP_111 55.500740ALA_112 0.502189GLY_113 11.908267ASN_114 107.208527ASN_115 78.811234GLY_116 41.453194MET_117 9.634291HIS_118 54.022118VAL_119 5.105174ALA_120 0.268693ASN_121 0.233495LEU_122 0.537387SER_123 4.004620LEU_124 21.927265GLY_125 55.952454SER_126 40.241180PRO_127 107.409439SER_128 57.988609PRO_129 85.021118SER_130 20.460915ALA_131 57.404362THR_132 74.438805LEU_133 12.091203GLU_134 73.382019GLN_135 114.870010ALA_136 2.122917VAL_137 1.074774ASN_138 55.622704SER_139 29.174965ALA_140 0.268693THR_141 27.962946SER_142 87.263145ARG_143 88.201218GLY_144 38.477882VAL_145 2.079151LEU_146 13.703363VAL_147 2.690253VAL_148 1.074774ALA_149 0.000000ALA_150 4.356600SER_151 0.000000GLY_152 12.628590ASN_153 84.248703SER_154 77.662354GLY_155 25.409861ALA_156 38.074570GLY_157 40.493744SER_158 53.915291ILE_159 4.352278SER_160 12.458543TYR_161 29.670284PRO_162 4.030401ALA_163 0.968253ARG_164 84.059120TYR_165 28.641129ALA_166 68.193314ASN_167 61.686481ALA_168 0.537387MET_169 0.586837ALA_170 0.000000VAL_171 0.000000GLY_172 0.000000ALA_173 0.933982THR_174 3.013133ASP_175 34.551376GLN_176 96.873039ASN_177 98.664368ASN_178 41.197159ASN_179 60.263512ARG_180 64.416336ALA_181 7.254722SER_182 91.590881PHE_183 52.126518SER_184 2.101459GLN_185 15.736279TYR_186 44.287792GLY_187 5.114592ALA_188 69.406563GLY_189 36.926083LEU_190 16.511177ASP_191 7.705349ILE_192 0.268693VAL_193 4.299094ALA_194 0.000000PRO_195 0.806080GLY_196 0.000000VAL_197 25.257177ASN_198 82.177422VAL_199 10.747736GLN_200 80.374527SER_201 2.008755THR_202 0.000000TYR_203 80.679886PRO_204 34.632195GLY_205 74.536827SER_206 74.964920THR_207 57.070065TYR_208 82.895500ALA_209 22.838940SER_210 69.045639LEU_211 49.708279ASN_212 86.905457GLY_213 2.686934THR_214 4.669909SER_215 15.225292MET_216 7.261287ALA_217 0.000000THR_218 0.000000PRO_219 0.806080HIS_220 2.662697VAL_221 0.268693ALA_222 0.000000GLY_223 0.000000ALA_224 7.206634ALA_225 1.039576ALA_226 0.268693LEU_227 1.074774VAL_228 1.541764LYS_229 39.262505GLN_230 54.501614LYS_231 81.154129ASN_232 30.004124PRO_233 91.917931SER_234 102.856705TRP_235 64.639481SER_236 51.797619ASN_237 24.866917VAL_238 78.458466GLN_239 73.981461ILE_240 14.474245ARG_241 41.242931ASN_242 64.644814HIS_243 50.671440LEU_244 5.127482LYS_245 48.820000ASN_246 115.264534THR_247 22.205376ALA_248 16.415077THR_249 60.503101SER_250 74.511597LEU_251 48.861599GLY_252 39.124340SER_253 49.811481THR_254 88.421982ASN_255 72.490181LEU_256 54.835758TYR_257 38.798912GLY_258 3.620916SER_259 35.017368GLY_260 0.537387LEU_261 8.598188VAL_262 4.519700ASN_263 16.763659ALA_264 3.413124GLU_265 37.942276ALA_266 15.871746ALA_267 3.947115THR_268 2.475746ARG_269 176.743362ION_270 0.000000ION_271 5.197493Subset REST:restmole.listSubset REST:SAVI8:E5-E15,E17-E18,E22,E38-E40,E42-E43,E73-E76,E82-E86,E103-E105,SAVI8:E108-E109,E111-E112,E115-E116,E122,E128-E144,E149-E150,E156-E157,SAVI8:E160-E162,E165-E168,E170-E171,E173,E180-E188,E190-E192,E200,SAVI8:E203-E204,E206,E211-E213,E215-E216,E227-E230,E255-E259,E261-E262,SAVI8:E267-E269restatom.listSubset REST:SAVI8:PRO E5:N,CD,CA,CG,CB,C,OSAVI8:TRP E6:N,CA,CD2,CE2,NE1,CD1,CG,CE3,CZ3,CH2,CZ2,CB,C,OSAVI8:GLY E7:N,CA,C,OSAVI8:ILE E8:N,CA,CD1,CG1,CB,CG2,C,OSAVI8:SER E9:N,CA,OG,CB,C,OSAVI8:ARG E10:N,CA,NH2,NH1,CZ,NE,CD,CG,CB,C,OSAVI8:VAL E11:N,CA,CG2,CG1,CB,C,OSAVI8:GLN E12:N,CA,NE2,OE1,CD,CG,CB,C,OSAVI8:ALA E13:N,CA,CB,C,OSAVI8:PRO E14:N,CD,CA,CG,CB,C,OSAVI8:ALA E15:N,CA,CB,C,OSAVI8:HIS E17:N,CA,CD2,NE2,CE1,ND1,CG,CB,C,OSAVI8:ASN E18:N,CA,ND2,OD1,CG,CB,C,OSAVI8:THR E22:N,CA,CG2,OG1,CB,C,OSAVI8:THR E38:N,CA,CG2,OG1,CB,C,OSAVI8:HIS E39:N,CA,CD2,NE2,CE1,ND1,CG,CB,C,OSAVI8:PRO E40:N,CD,CA,CG,CB,C,OSAVI8:LEU E42:N,CA,CD2,CD1,CG,CB,C,OSAVI8:ASN E43:N,CA,ND2,OD1,CG,CB,C,OSAVI8:ALA E73:N,CA,CB,C,OSAVI8:ALA E74:N,CA,CB,C,OSAVI8:LEU E75:N,CA,CD2,CD1,CG,CB,C,OSAVI8:ASN E76:N,CA,ND2,OD1,CG,CB,C,OSAVI8:LEU E82:N,CA,CD2,CD1,CG,CB,C,OSAVI8:GLY E83:N,CA,C,OSAVI8:VAL E84:N,CA,CG2,CG1,CB,C,OSAVI8:ALA E85:N,CA,CB,C,OSAVI8:PRO E86:N,CD,CA,CG,CB,C,OSAVI8:SER E103:N,CA,OG,CB,C,OSAVI8:VAL E104:N,CA,CG2,CG1,CB,C,OSAVI8:SER E105:N,CA,OG,CB,C,OSAVI8:ALA E108:N,CA,CB,C,OSAVI8:GLN E109:N,CA,NE2,OE1,CD, CG,CB,C,OSAVI8:LEU E111:N,CA,CD2,CD1,CG,CB,C,OSAVI8:GLU E112:N,CA,OE2,OE1,CD,CG,CB,C,OSAVI8:GLY E115:N,CA,C,OSAVI8:ASN E116:N,CA,ND2,OD1,CG,CB,C,OSAVI8:ALA E122:N,CA,CB,C,OSAVI8:SER E128:N,CA,OG,CB,C,OSAVI8:PRO E129:N,CD,CA,CG,CB,C,OSAVI8:SER E130:N,CA,OG,CB,C,OSAVI8:PRO E131:N,CD,CA,CG,CB,C,OSAVI8:SER E132:N,CA,OG,CB,C,OSAVI8:ALA E133:N,CA,CB,C,OSAVI8:THR E134:N,CA,CG2,OG1,CB,C,OSAVI8:LEU E135:N,CA,CD2,CD1,CG,CB,C,OSAVI8:GLU E136:N,CA,OE2,OE1,CD,CG,CB,C,OSAVI8:GLN E137:N,CA,NE2,OE1,CD, CG,CB,C,OSAVI8:ALA E138:N,CA,CB,C,OSAVI8:VAL E139:N,CA,CG2,CG1,CB,C,OSAVI8:ASN E140:N,CA,ND2,OD1,CG, CB,C,OSAVI8:SER E141:N,CA,OG,CB,C,OSAVI8:ALA E142:N,CA,CB,C,OSAVI8:THR E143:N,CA,CG2,OG1,CB,C,OSAVI8:SER E144:N,CA,OG,CB,C,OSAVI8:VAL E149:N,CA,CG2,CG1,CB,C,OSAVI8:VAL E150:N,CA,CG2,CG1,CB,C,OSAVI8:SER E156:N,CA,OG,CB,C,OSAVI8:GLY E157:N,CA,C,OSAVI8:ALA E160:N,CA,CB,C,OSAVI8:GLY E161:N,CA,C,OSAVI8:SER E162:N,CA,OG,CB,C,OSAVI8:ILE E165:N,CA,CD1,CG1,CB,CG2,C,OSAVI8:SER E166:N,CA,OG,CB,C,OSAVI8:TYR E167:N,CA,OH,CZ,CD2,CE2,CE1,CD1,CG,CB,C,OSAVI8:PRO E168:N,CD,CA,CG,CB,C,OSAVI8:ARG E170:N,CA,NH2,NH1,CZ,NE,CD,CG,CB,C,OSAVI8:TYR E171:N,CA,OH,CZ,CD2,CE2,CE1,CD1,CG,CB,C,OSAVI8:ASN E173:N,CA,ND2,OD1,CG,CB,C,OSAVI8:THR E180:N,CA,CG2,OG1,CB,C,OSAVI8:ASP E181:N,CA,OD2,OD1,CG,CB,C,OSAVI8:GLN E182:N,CA,NE2,OE1,CD,CG,CB,C,OSAVI8:ASN E183:N,CA,ND2,OD1,CG,CB,C,OSAVI8:ASN E184:N,CA,ND2,OD1,CG,CB,C,OSAVI8:ASN E185:N,CA,ND2,OD1,CG,CB,C,OSAVI8:ARG E186:N,CA,NH2,NH1,CZ,NE,CD,CG,CB,C,OSAVI8:ALA E187:N,CA,CB,C,OSAVI8:SER E188:N,CA,OG,CB,C,OSAVI8:SER E190:N,CA,OG,CB,C,OSAVI8:GLN E191:N,CA,NE2,OE1,CD,CG,CB,C,OSAVI8:TYR E192:N,CA,OH,CZ,CD2,CE2,CE1,CD1,CG,CB,C,OSAVI8:ALA E200:N,CA,CB,C,OSAVI8:VAL E203:N,CA,CG2,CG1,CB,C,OSAVI8:ASN E204:N,CA,ND2,OD1,CG,CB,C,OSAVI8:GLN E206:N,CA,NE2,OE1,CD,CG, CB,C,OSAVI8:GLY E211:N,CA,C,OSAVI8:SER E212:N,CA,OG,CB,C,OSAVI8:THR E213:N,CA,CG2,OG1,CB,C,OSAVI8:ALA E215:N,CA,CB,C,OSAVI8:SER E216:N,CA,OG,CB,C,OSAVI8:VAL E227:N,CA,CG2,CG1,CB,C,OSAVI8:ALA E228:N,CA,CB,C,OSAVI8:GLY E229:N,CA,C,OSAVI8:ALA E230:N,CA,CB,C,OSAVI8:THR E255:N,CA,CG2,OG1,CB,C,OSAVI8:SER E256:N,CA,OG,CB,C,OSAVI8:LEU E257:N,CA,CD2,CD1,CG,CB,C,OSAVI8:GLY E258:N,CA,C,OSAVI8:SER E259:N,CA,OG,CB,C,OSAVI8:ASN E261:N,CA,ND2,OD1,CG,CB,C,OSAVI8:LEU E262:N,CA,CD2,CD1,CG,CB,C,OSAVI8:LEU E267:N,CA,CD2,CD1,CG,CB,C,OSAVI8:VAL E268:N,CA,CG2,CG1,CB,C,OSAVI8:ASN E269:N,CA,ND2,OD1,CG,CB,C,OSubset SUB5B:sub5bmole.listSubset SUB5B:SAVI8:E2-E4,E16,E19-E21,E23-E24,E28,E37,E41,E44-E45,E77-E81,E87-E88,SAVI8:E90,E113-E114,E117-E118,E120-E121,E145-E148,E169,E172,E174-E176,SAVI8:E193-E196,E198-E199,E214,E231-E234,E236,E243,E247,E250,E253-E254,SAVI8:E260,E263-E266,E270-E273,M276H-M277Hsub5batom.listSubset SUB5B:SAVI8:GLN E2:N,CA,NE2,OE1,CD,CG,CB,C,OSAVI8:SER E3:N,CA,OG,CB,C,OSAVI8:VAL E4:N,CA,CG2,CG1,CB,C,OSAVI8:ALA E16:N,CA,CB,C,OSAVI8:ARG E19:N,CA,NH2,NH1,CZ,NE,CD,CG,CB,C,OSAVI8:GLY E20:N,CA,C,OSAVI8:LEU E21:N,CA,CD2,CD1,CG,CB,C,OSAVI8:GLY E23:N,CA,C,OSAVI8:SER E24:N,CA,OG,CB,C,OSAVI8:VAL E28:N,CA,CG2,CG1,CB,C,OSAVI8:SER E37:N,CA,OG,CB,C,OSAVI8:ASP E41:N,CA,OD2,OD1,CG,CB,C,OSAVI8:ILE E44:N,CA,CD1,CG1,CB,CG2,C,OSAVI8:ARG E45:N,CA,NH2,NH1,CZ,NE,CD,CG,CB,C,OSAVI8:ASN E77:N,CA,ND2,OD1,CG,CB,C,OSAVI8:SER E78:N,CA,OG,CB,C,OSAVI8:ILE E79:N,CA,CD1,CG1,CB,CG2,C,OSAVI8:GLY E80:N,CA,C,OSAVI8:VAL E81:N,CA,CG2,CG1,CB,C,OSAVI8:SER E87:N,CA,OG,CB,C,OSAVI8:ALA E88:N,CA,CB,C,OSAVI8:LEU E90:N,CA,CD2,CD1,CG,CB,C,OSAVI8:TRP E113:N,CA,CD2,CE2,NE1,CD1,CG,CE3,CZ3,CH2,CZ2,CB,C,OSAVI8:ALA E114:N,CA,CB,C,OSAVI8:ASN E117:N,CA,ND2,OD1,CG,CB,C,OSAVI8:GLY E118:N,CA,C,OSAVI8:HIS E120:N,CA,CD2,NE2,CE1,ND1,CG,CB,C,OSAVI8:VAL E121:N,CA,CG2,CG1,CB,C,OSAVI8:ARG E145:N,CA,NH2,NH1,CZ,NE,CD,CG,CB,C,OSAVI8:GLY E146:N,CA,C,OSAVI8:VAL E147:N,CA,CG2,CG1,CB,C,OSAVI8:LEU E148:N,CA,CD2,CD1,CG,CB,C,OSAVI8:ALA E169:N,CA,CB,C,OSAVI8:ALA E172:N,CA,CB,C,OSAVI8:ALA E174:N,CA,CB,C,OSAVI8:MET E175:N,CA,CE,SD,CG,CB,C,OSAVI8:ALA E176:N,CA,CB,C,OSAVI8:GLY E193:N,CA,C,OSAVI8:ALA E194:N,CA,CB,C,OSAVI8:GLY E195:N,CA,C,OSAVI8:LEU E196:N,CA,CD2,CD1,CG,CB,C,OSAVI8:ILE E198:N,CA,CD1,CG1,CB,CG2,C,OSAVI8:VAL E199:N,CA,CG2,CG1,CB,C,OSAVI8:TYR E214:N,CA,OH,CZ,CD2,CE2,CE1,CD1,CG,CB,C,OSAVI8:ALA E231:N,CA,CB,C,OSAVI8:ALA E232:N,CA,CB,C,OSAVI8:LEU E233:N,CA,CD2,CD1,CG,CB,C,OSAVI8:VAL E234:N,CA,CG2,CG1,CB,C,OSAVI8:GLN E236:N,CA,NE2,OE1,CD,CG,CB,C,OSAVI8:ASN E243:N,CA,ND2,OD1,CG,CB,C,OSAVI8:ARG E247:N,CA,NH2,NH1,CZ,NE,CD,CG,CB,C,OSAVI8:LEU E250:N,CA,CD2,CD1,CG,CB,C,OSAVI8:THR E253:N,CA,CG2,OG1,CB,C,OSAVI8:ALA E254:N,CA,CB,C,OSAVI8:THR E260:N,CA,CG2,OG1,CB,C,OSAVI8:TYR E263:N,CA,OH,CZ,CD2,CE2,CE1,CD1,CG,CB,C,OSAVI8:GLY E264:N,CA,C,OSAVI8:SER E265:N,CA,OG,CB,C,OSAVI8:GLY E266:N,CA,C,OSAVI8:ALA E270:N,CA,CB,C,OSAVI8:GLU E271:N,CA,OE2,OE1,CD,CG,CB,C,OSAVI8:ALA E272:N,CA,CB,C,OSAVI8:ALA E273:N,CA,CB,C,OSAVI8:ION M276H:CASAVI8:ION M277H:CASubset ACTSITE:actsitemole.listSubset ACTSITE:SAVI8:E29-E35,E48-E51,E54,E58-E72,E91-E102,E106-E107,E110,E123-E127,SAVI8:E151-E155,E177-E179,E189,E201-E202,E205,E207-E210,E217-E226actsiteatom.listSubset ACTSITE:
SAVI8:ALA E29:N,CA,CB,C,O
SAVI8:VAL E30:N,CA,CG2,CG1,CB,C,O
SAVI8:LEU E31:N,CA,CD2,CD1,CG,CB,C,O
SAVI8:ASP E32:N,CA,OD2,OD1,CG,CB,C,O
SAVI8:THR E33:N,CA,CG2,OG1,CB,C,O
SAVI8:GLY E34:N,CA,C,O
SAVI8:ILE E35:N,CA,CD1,CG1,CB,CG2,C,O
SAVI8:ALA E48:N,CA,CB,C,O
SAVI8:SER E49:N,CA,OG,CB,C,O
SAVI8:PHE E50:N,CA,CD2,CE2,CZ,CE1,CD1,CG,CB,C,O
SAVI8:VAL E51:N,CA,CG2,CG1,CB,C,O
SAVI8:GLU E54:N,CA,OE2,OE1,CD,CG,CB,C,O
SAVI8:THR E58:N,CA,CG2,OG1,CB,C,O
SAVI8:GLN E59:N,CA,NE2,OE1,CD,CG,CB,C,O
SAVI8:ASP E60:N,CA,OD2,OD1,CG,CB,C,O
SAVI8:GLY E61:N,CA,C,O
SAVI8:ASN E62:N,CA,ND2,OD1,CG,CB,C,O
SAVI8:GLY E63:N,CA,C,O
SAVI8:HIS E64:N,CA,CD2,NE2,CE1,ND1,CG,CB,C,O
SAVI8:GLY E65:N,CA,C,O
SAVI8:THR E66:N,CA,CG2,OG1,CB,C,O
SAVI8:HIS E67:N,CA,CD2,NE2,CE1,ND1,CG,CB,C,O
SAVI8:VAL E68:N,CA,CG2,CG1,CB,C,O
SAVI8:ALA E69:N,CA,CB,C,O
SAVI8:GLY E70:N,CA,C,O
SAVI8:THR E71:N,CA,CG2,OG1,CB,C,O
SAVI8:ILE E72:N,CA,CD1,CG1,CB,CG2,C,O
SAVI8:TYR E91:N,CA,OH,CZ,CD2,CE2,CE1,CD1,CG,CB,C,O
SAVI8:ALA E92:N,CA,CB,C,O
SAVI8:VAL E93:N,CA,CG2,CG1,CB,C,O
SAVI8:LYS E94:N,CA,NZ,CE,CD,CG,CB,C,O
SAVI8:VAL E95:N,CA,CG2,CG1,CB,C,O
SAVI8:LEU E96:N,CA,CD2,CD1,CG,CB,C,O
SAVI8:GLY E97:N,CA,C,O
SAVI8:ALA E98:N,CA,CB,C,O
SAVI8:SER E99:N,CA,OG,CB,C,O
SAVI8:GLY E100:N,CA,C,O
SAVI8:SER E101:N,CA,OG,CB,C,O
SAVI8:GLY E102:N,CA,C,O
SAVI8:SER E106:N,CA,OG,CB,C,O
SAVI8:ILE E107:N,CA,CD1,CG1,CB,CG2,C,O
SAVI8:GLY E110:N,CA,C,O
SAVI8:ASN E123:N,CA,ND2,OD1,CG,CB,C,O
SAVI8:LEU E124:N,CA,CD2,CD1,CG,CB,C,O
SAVI8:SER E125:N,CA,OG,CB,C,O
SAVI8:LEU E126:N,CA,CD2,CD1,CG,CB,C,O
SAVI8:GLY E127:N,CA,C,O
SAVI8:ALA E151:N,CA,CB,C,O
SAVI8:ALA E152:N,CA,CB,C,O
SAVI8:SER E153:N,CA,OG,CB,C,O
SAVI8:GLY E154:N,CA,C,O
SAVI8:ASN E155:N,CA,ND2,OD1,CG,CB,C,O
SAVI8:VAL E177:N,CA,CG2,CG1,CB,C,O
SAVI8:GLY E178:N,CA,C,O
SAVI8:ALA E179:N,CA,CB,C,O
SAVI8:PHE E189:N,CA,CD2,CE2,CZ,CE1,CD1,CG,CB,C,O
SAVI8:PRO E201:N,CD,CA,CG,CB,C,O
SAVI8:GLY E202:N,CA,C,O
SAVI8:VAL E205:N,CA,CG2,CG1,CB,C,O
SAVI8:SER E207:N,CA,OG,CB,C,O
SAVI8:THR E208:N,CA,CG2,OG1,CB,C,O
SAVI8:TYR E209:N,CA,OH,CZ,CD2,CE2,CE1,CD1,CG,CB,C,O
SAVI8:PRO E210:N,CD,CA,CG,CB,C,O
SAVI8:LEU E217:N,CA,CD2,CD1,CG,CB,C,O
SAVI8:ASN E218:N,CA,ND2,OD1,CG,CB,C,O
SAVI8:GLY E219:N,CA,C,O
SAVI8:THR E220:N,CA,CG2,OG1,CB,C,O
SAVI8:SER E221:N,CA,OG,CB,C,O
SAVI8:MET E222:N,CA,CE,SD,CG,CB,C,O
SAVI8:ALA E223:N,CA,CB,C,O
SAVI8:THR E224:N,CA,CG2,OG1,CB,C,O
SAVI8:PRO E225:N,CD,CA,CG,CB,C,O
SAVI8:HIS E226:N,CA,CD2,NE2,CE1,ND1,CG,CB,C,OSubset RESTx:restxmole.listSubset RESTX:
NEWMODEL:E5,E13-E14,E22,E38-E40,
E42,E73-E76,E82-E86,E103-E105,
NEWMODEL:E108,E122,E133-E135,E137-E140,
E149-E150,E173,E204,E206,
NEWMODEL:E211-E213,E215-E216,E227- E229,
E258,E269restxatom.listSubset RESTX:
NEWMODEL:PRO E5:N,CD,CA,CG,CB,C,O
NEWMODEL:ALA E13:N,CA,CB,C,O
NEWMODEL:PRO E14:N,CD,CA,CG,CB,C,O
NEWMODEL:THR E22:N,CA,CG2,OG1,CB,C,O
NEWMODEL:THR E38:N,CA,CG2,OG1,CB,C,O
NEWMODEL:HIS E39:N,CA,CD2,NE2,CE1,ND1,CG,CB,C,O
NEWMODEL:PRO E40:N,CD,CA,CG,CB,C,O
NEWMODEL:LEU E42:N,CA,CD2,CD1,CG,CB,C,O
NEWMODEL:ALA E73:N,CA,CB,C,O
NEWMODEL:ALA E74:N,CA,CB,C,O
NEWMODEL:LEU E75:N,CA,CD2,CD1,CG,CB,C,O
NEWMODEL:ASN E76:N,CA,ND2,OD1,CG,CB,C,O
NEWMODEL:LEU E82:N,CA,CD2,CD1,CG,CB,C,O
NEWMODEL:GLY E83:N,CA,C,O
NEWMODEL:VAL E84:N,CA,CG2,CG1,CB,C,O
NEWMODEL:ALA E85:N,CA,CB,C,O
NEWMODEL:PRO E86:N,CD,CA,CG,CB,C,O
NEWMODEL:SER E103:N,CA,OG,CB,C,O
NEWMODEL:VAL E104:N,CA,CG2,CG1,CB,C,O
NEWMODEL:SER E105:N,CA,OG,CB,C,O
NEWMODEL:ALA E108:N,CA,CB,C,O
NEWMODEL:ALA E122:N,CA,CB,C,O
NEWMODEL:ALA E133:N,CA,CB,C,O
NEWMODEL:THR E134:N,CA,CG2,OG1,CB,C,O
NEWMODEL:LEU E135:N,CA,CD2,CD1,CG,CB,C,ONEWMODEL:GLN E137:N,CA,NE2,OE1,CD,CG,CB,C,ONEWMODEL:ALA E138:N,CA,CB,C,ONEWMODEL:VAL E139:N,CA,CG2,CG1,CB,C,ONEWMODEL:ASN E140:N,CA,ND2,OD1,CG,CB,C,ONEWMODEL:VAL E149:N,CA,CG2,CG1,CB,C,ONEWMODEL:VAL E150:N,CA,CG2,CG1,CB,C,ONEWMODEL:ASN E173:N,CA,ND2,OD1,CG,CB,C,ONEWMODEL:ASN E204:N,CA,ND2,OD1,CG,CB,C,ONEWMODEL:GLN E206:N,CA,NE2,OE1,CD,CG,CB,C,ONEWMODEL:GLY E211:N,CA,C,ONEWMODEL:SER E212:N,CA,OG,CB,C,ONEWMODEL:THR E213:N,CA,CG2,OG1,CB,C,ONEWMODEL:ALA E215:N,CA,CB,C,ONEWMODEL:SER E216:N,CA,OG,CB,C,ONEWMODEL:VAL E227:N,CA,CG2,CG1,CB,C,ONEWMODEL:ALA E228:N,CA,CB,C,ONEWMODEL:GLY E229:N,CA,C,ONEWMODEL:GLY E258:N,CA,C,ONEWMODEL:ASN E269:N,CA,ND2,OD1,CG,CB,C,O实施例3:PD498中适于添加羧酸连接基团(-COOH)的合适取代
按照实施例1所述建立PD498的3D结构。
按照下述找出添加羧基连接基团(天冬氨酸和谷氨酸)的合适位置。遵循实施例1所述的操作。Insight(BIOSYM)中运行的各个指令示于下述指令文件makeDEzone.bcl和makeDEzone2.bcl中:
保守性取代:makeDEzone.bclDelete Subset*Color Molecule Atoms * Specified Specification 255,0,255Zone Subset ASP:asp:od* Static monomer/residue 10 Color_Subset255,255,0Zone Subset GLU:glu:oe* Static monomer/residue 10 Color_Subset255,255,0#NOTE:editnextline C-terminal residue number according to theproteinzone Subset CTERM:280:O Static monomer/residue 10 Color_Subset255,255,0#NOTE:editnextline ACTSITE residues according to the proteinZone Subset ACTSITE:39,72,226 Static monomer/residue 8Color_Subset 255,255,0Combine Subset ALLZONE Union ASP GLUCombine Subset ALLZONE Union ALLZONE CTERMCombine Subset ALLZONE Union ALLZONE ACTSITE#NOTE:editnextline object name according to the proteinCombine Subset REST Difference PD498FINALMODEL ALLZONEList Subset REST Atom Output_File restatom.listList Subset REST monomer/residue Output_File restmole.listColor Molecule Atoms ACTSITE Specified Specification 255,0,0List Subset ACTSITE Atom Output_File actsiteatom.listList Subset ACTSITE monomer/residue Output_Fileactsitemole.list#Zone Subset REST5A REST Static Monomer/Residue 5 -Color_SubsetCombine Subset SUB5A Difference REST5A ACTSITECombine Subset SUB5B Difference SUB5A RESTColor Molecule Atoms SUB5B Specified Specification 255,255,255List Subset SUB5B Atom Output_File sub5batom.listList Subset SUB5B monomer/residue Output_File sub5bmole.list#Now identify sites for asn→asp & gln→glu substitutions and...#continue with makezone2.bcl.#Use grep command to identify asn/gln in restatom.list ...#sub5batom.list & accsiteatom.list说明:
REST亚组包含Gln33和Asn245,SUB5B包含Gln12,Gln126、Asn209,Gln242,Asn246,Gln248和Asn266,所有这些残基都是溶剂暴露型的。
鉴定出PD498内Q12E或Q12D、Q33E或Q33D、Q126E或Q126D、N209D或N209E、Q242E或Q242D、N245D或N245E、N246D或N246E、Q248E或Q248D以及N266D或N266E这些取代是进行本发明范围内诱变的一些位点。将在下面第2部分中进行残基取代,并进一步分析:
非保守性取代:makeDEzone2.bcl#sourcefile makezone2.bcl Claus von der Osten 961128##having scanned lists(grep gln/asn command)and identifiedsites for ...#asn→asp & gln→glu substitutions#NOTE:editnextline object name according to proteinCopy object -To_Clipboard -Displace PD498FINALMODEL newmodelBiopolymer#NOTE:editnextline object name according to proteinBlank Object On PD498FINALMODEL#NOTE:editnextlines with asn→asp & gln→glu positionsReplace Residue newmodel:33 glu LReplace Residue newmodel:245 asp LReplace Residue newmodel:12 glu LReplace Residue newmodel:126 glu LReplace Residue newmodel:209 asp LReplace Residue newmodel:242 glu LReplace Residue newmodel:246 asp LReplace Residue newmodel:248 glu LReplace Residue newmodel:266 asp L##Now repeat analysis done prior to asn→asp & gln→glu,...#now including introduced asp & gluColor Molecule Atoms newmodel Specified specification 255,0,255Zone Subset ASPx newmodel:asp:od* Static monomer/residue 10Color_Subset 255,255,0Zone Subset GLUx newmodel:glu:oe* Static monomer/residue 10Color_Subset 255,255,0#NOTE:editnextline C-terminal residue number according to theproteinZone Subset CTERMx newmodel:280:O Static monomer/residue 10Color_Subset 255,255,0#NOTE:editnextline ACTSITEx residues according to the proteinZone Subset ACTSITEx newmodel:39,72,226 Static monomer/residue8 Color_Subset 255,255,0Combine_Subset ALLZONEx Union ASPx GLUxCombine Subset ALLZONEx Union ALLZONEx CTERMxCombine Subset ALLZONEx Union ALLZONEx ACTSITExCombine Subset RESTx Difference newmodel ALLZONExList Subset RESTx Atom Output_File restxatom.listList Subset RESTx monomer/residue Output_File restxmole.list#Color Molecule Atoms ACTSITEx Specified Specification 255,0,0List Subset ACTSITEx Atom Output_File actsitexatom.listList Subset ACTSITEx monomer/residue Output_Fileactsitexmole.list##read restxatom.list or restxmole.list to identify sites for(not_gluasp)→gluasp ...#subst.if needed说明:
RESTx亚组仅含有两个残基:A233和G234,它们都不是溶剂暴露型的。不需要进一步诱变来达到对表面的完全保护。然而,有可能需要除去活性位点区域内的一些反应活性羧基,以确保能到达PD498的活性位点。ACTSITE亚组内的酸性残基是:D39、D58、D68和D106。这些残基中,仅后面两个残基是溶剂暴露型的,且D39是一个功能性残基。根据本发明,D68N、D68Q、D106N和D106Q是比较合适的突变。
实施例3的有关数据:
PD498MODEL的溶剂可及性数据:参见上述实施例1。Subset REST:restmole.listSubset REST:
PD498FINALMODEL:10-11,33-35,54-55,129-130,
221,233-234,236,240,243,
PD498FINALMODEL:245,262,264-265 restatom.listSubset REST:PD498FINALMODEL:ALA 10:N,CA,C,O,CBPD498FINALMODEL:TYR 11:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OHPD498FINALMODEL:GLN 33:N,CA,C,O,CB,CG,CD,OE1,NE2PD498FINALMODEL:THR 34:N,CA,C,O,CB,OG1,CG2PD498FINALMODEL:VAL 35:N,CA,C,O,CB,CG1,CG2PD498FINALMODEL:ILE 54:N,CA,C,O,CB,CG1,CG2,CD1PD498FINALMODEL:LYS 55:N,CA,C,O,CB,CG,CD,CE,NZPD498FINALMODEL:LYS 129:N,CA,C,O,CB,CG,CD,CE,NZPD498FINALMODEL:VAL 130:N,CA,C,O,CB,CG1,CG2PD498FINALMODEL:TYR 221:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OHPD498FINALMODEL:ALA 233:N,CA,C,O,CBPD498FINALMODEL:GLY 234:N,CA,C,OPD498FINALMODEL:ALA 236:N,CA,C,O,CBPD498FINALMODEL:ALA 240:N,CA,C,O,CBPD498FINALMODEL:GLY 243:N,CA,C,OPD498FINALMODEL:ASN 245:N,CA,C,O,CB,CG,OD1,ND2PD498FINALMODEL:GLY 262:N,CA,C,OPD498FINALMODEL:GLY 264:N,CA,C,OPD498FINALMODEL:THR 265:N,CA,C,O,CB,OG1,CG2Subset SUB5B:sub5bmole.listSubset SUB5B:PD498FINALMODEL:6-9,12-13,31-32,51-53,56,81,93-94,97-99,122,126-128,PD498FINALMODEL:131,155-157,159,197-199,209,211,219-220,232,235,PD498FINALMODEL:237-239,241-242,244,246-249,253,260-261,263,266-268sub5batom.list
Subset SUB5B:PD498FINALMODEL:PRO 6:N,CA,CD,C,O,CB,CGPD498FINALMODEL:TYR 7:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OHPD498FINALMODEL:TYR 8:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OHPD498FINALMODEL:SER 9:N,CA,C,O,CB,OGPD498FINALMODEL:GLN 12:N,CA,C,O,CB,CG,CD,OE1,NE2PD498FINALMODEL:TYR 13:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OHPD498FINALMODEL:SER 31:N,CA,C,O,CB,OGPD498FINALMODEL:THR 32:N,CA,C,O,CB,OG1,CG2PD498FINALMODEL:ARG 51:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2PD498FINALMODEL:LYS 52:N,CA,C,O,CB,CG,CD,CE,NZPD498FINALMODEL:VAL 53:N,CA,C,O,CB,CG1,CG2PD498FINALMODEL:GLY 56:N,CA,C,OPD498FINALMODEL:ALA 81:N,CA,C,O,CBPD498FINALMODEL:MET 93:N,CA,C,O,CB,CG,SD,CEPD498FINALMODEL:ALA 94:N,CA,C,O,CBPD498FINALMODEL:THR 97:N,CA,C,O,CB,OG1,CG2PD498FINALMODEL:LYS 98:N,CA,C,O,CB,CG,CD,CE,NZPD498FINALMODEL:ILE 99:N,CA,C,O,CB,CG1,CG2,CD1PD498FINALMODEL:TYR 122:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OHPD498FINALMODEL:GLN 126:N,CA,C,O,CB,CG,CD,OE1,NE2PD498FINALMODEL:GLY 127:N,CA,C,OPD498FINALMODEL:ALA 128:N,CA,C,O,CBPD498FINALMODEL:LEU 131:N,CA,C,O,CB,CG,CD1,CD2PD498FINALMODEL:GLY 155:N,CA,C,OPD498FINALMODEL:ALA 156:N,CA,C,O,CBPD498FINALMODEL:VAL 157:N,CA,C,O,CB,CG1,CG2PD498FINALMODEL:VAL 159:N,CA,C,O,CB,CG1,CG2PD498FINALMODEL:TYR 197:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OHPD498FINALMODEL:GLY 198:N,CA,C,OPD498FINALMODEL:THR 199:N,CA,C,O,CB,OG1,CG2PD498FINALMODEL:ASN 209:N,CA,C,O,CB,CG,OD1,ND2PD498FINALMODEL:ALA 211:N,CA,C,O,CBPD498FINALMODEL:TYR 219:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OHPD498FINALMODEL:SER 220:N,CA,C,O,CB,OGPD498FINALMODEL:VAL 232:N,CA,C,O,CB,CG1,CG2PD498FINALMODEL:LEU 235:N,CA,C,O,CB,CG,CD1,CD2PD498FINALMODEL:ALA 237:N,CA,C,O,CBPD498FINALMODEL:LEU 238:N,CA,C,O,CB,CG,CD1,CD2PD498FINALMODEL:LEU 239:N,CA,C,O,CB,CG,CD1,CD2PD498FINALMODEL:SER 241:N,CA,C,O,CB,OGPD498FINALMODEL:GLN 242:N,CA,C,O,CB,CG,CD,OE1,NE2PD498FINALMODEL:LYS 244:N,CA,C,O,CB,CG,CD,CE,NZPD498FINALMODEL:ASN 246:N,CA,C,O,CB,CG,OD1,ND2PD498FINALMODEL:VAL 247:N,CA,C,O,CB,CG1,CG2PD498FINALMODEL:GLN 248:N,CA,C,O,CB,CG,CD,OE1,NE2PD498FINALMODEL:ILE 249:N,CA,C,O,CB,CG1,CG2,CD1PD498FINALMODEL:ILE 253:N,CA,C,O,CB,CG1,CG2,CD1PD498FINALMODEL:ILE 260:N,CA,C,O,CB,CG1,CG2,CD1PD498FINALMODEL:SER 261:N,CA,C,O,CB,OGPD498FINALMODEL:THR 263:N,CA,C,O,CB,OG1,CG2PD498FINALMODEL:ASN 266:N,CA,C,O,CB,CG,OD1,ND2PD498FINALMODEL:PHE 267:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZPD498FINALMODEL:LYS 268:N,CA,C,O,CB,CG,CD,CE,NZSubset ACTSITE:actsitemole.listSubset ACTSITE:
PD498FINALMODEL:36-42,57-60,66-80,100-110,
115-116,119,132-136,160-164,
PD498FINALMODEL:182-184,194,206-207,210,
212-215,222-231actsiteatom.listSubset ACTSITE:
PD498FINALMODEL:ALA 36:N,CA,C,O,CB
PD498FINALMODEL:VAL 37:N,CA,C,O,CB,CG1,CG2
PD498FINALMODEL:LEU 38:N,CA,C,O,CB,CG,CD1,CD2
PD498FINALMODEL:ASP 39:N,CA,C,O,CB,CG,OD1,OD2
PD498FINALMODEL:SER 40:N,CA,C,O,CB,OG
PD498FINALMODEL:GLY 41:N,CA,C,O
PD498FINALMODEL:VAL 42:N,CA,C,O,CB,CG1,CG2
PD498FINALMODEL:TYR
57:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH
PD498FINALMODEL:ASP 58:N,CA,C,O,CB,CG,OD1,OD2
PD498FINALMODEL:PHE
59:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ
PD498FINALMODEL:ILE 60:N,CA,C,O,CB,CG1,CG2,CD1
PD498FINALMODEL:PRO 66:N,CA,CD,C,O,CB,CG
PD498FINALMODEL:MET 67:N,CA,C,O,CB,CG,SD,CE
PD498FINALMODEL:ASP 68:N,CA,C,O,CB,CG,OD1,OD2
PD498FINALMODEL:LEU 69:N,CA,C,O,CB,CG,CD1,CD2
PD498FINALMODEL:ASN 70:N,CA,C,O,CB,CG,OD1,ND2
PD498FINALMODEL:GLY 71:N,CA,C,OPD498FINALMODEL:HIS 72:N,CA,C,O,CB,CG,ND1,CD2,CE1,NE2PD498FINALMODEL:GLY 73:N,CA,C,OPD498FINALMODEL:THR 74:N,CA,C,O,CB,OG1,CG2PD498FINALMODEL:HIS 75:N,CA,C,O,CB,CG,ND1,CD2,CE1,NE2PD498FINALMODEL:VAL 76:N,CA,C,O,CB,CG1,CG2PD498FINALMODEL:ALA 77:N,CA,C,O,CBPD498FINALMODEL:GLY 78:N,CA,C,OPD498FINALMODEL:THR 79:N,CA,C,O,CB,OG1,CG2PD498FINALMODEL:VAL 80:N,CA,C,O,CB,CG1,CG2PD498FINALMODEL:LEU 100:N,CA,C,O,CB,CG,CD1,CD2PD498FINALMODEL:ALA 101:N,CA,C,O,CBPD498FINALMODEL:VAL 102:N,CA,C,O,CB,CG1,CG2PD498FINALMODEL:ARG 103:N,CA,C,O,CB,
CG,CD,NE,CZ,NH1,NH2PD498FINALMODEL:VAL 104:N,CA,C,O,CB,CG1,CG2PD498FINALMODEL:LEU 105:N,CA,C,O,CB,CG,CD1,CD2PD498FINALMODEL:ASP 106:N,CA,C,O,CB,CG,OD1,OD2PD498FINALMODEL:ALA 107:N,CA,C,O,CBPD498FINALMODEL:ASN 108:N,CA,C,O,CB,CG,OD1,ND2PD498FINALMODEL:GLY 109:N,CA,C,OPD498FINALMODEL:SER 110:N,CA,C,O,CB,OGPD498FINALMODEL:SER 115:N,CA,C,O,CB,OGPD498FINALMODEL:ILE 116:N,CA,C,O,CB,
CG1,CG2,CD1PD498FINALMODEL:GLY 119:N,CA,C,OPD498FINALMODEL:ASN 132:N,CA,C,O,CB,CG,OD1,ND2PD498FINALMODEL:LEU 133:N,CA,C,O,CB,CG,CD1,CD2PD498FINALMODEL:SER 134:N,CA,C,O,CB,OGPD498FINALMODEL:LEU 135:N,CA,C,O,CB,CG,CD1,CD2PD498FINALMODEL:GLY 136:N,CA,C,OPD498FINALMODEL:ALA 160:N,CA,C,O,CBPD498FINALMODEL:ALA 161:N,CA,C,O,CBPD498FINALMODEL:ALA 162:N,CA,C,O,CBPD498FINALMODEL:GLY 163:N,CA,C,OPD498FINALMODEL:ASN 164:N,CA,C,O,CB,CG,OD1,ND2PD498FINALMODEL:VAL 182:N,CA,C,O,CB,CG1,CG2PD498FINALMODEL:GLY 183:N,CA,C,OPD498FINALMODEL:ALA 184:N,CA,C,O,CBPD498FINALMODEL:PHE 194:N,CA,C,O,CB,
CG,CD1,CD2,CE1,CE2,CZPD498FINALMODEL:PRO 206:N,CA,CD,C,O,CB,CGPD498FINALMODEL:GLY 207:N,CA,C,OPD498FINALMODEL:ILE 210:N,CA,C,O,CB,
CG1,CG2,CD1PD498FINALMODEL:SER 212:N,CA,C,O,CB,OGPD498FINALMODEL:THR 213:N,CA,C,O,CB,OG1,CG2PD498FINALMODEL:VAL 214:N,CA,C,O,CB,CG1,CG2PD498FINALMODEL:PRO 215:N,CA,CD,C,O,CB,CGPD498FINALMODEL:MET 222:N,CA,C,O,CB,CG,SD,CEPD498FINALMODEL:SER 223:N,CA,C,O,CB,OGPD498FINALMODEL:GLY 224:N,CA,C,OPD498FINALMODEL:THR 225:N,CA,C,O,CB,OG1,CG2PD498FINALMODEL:SER 226:N,CA,C,O,CB,OGPD498FINALMODEL:MET 227:N,CA,C,O,CB,CG,SD,CEPD498FINALMODEL:ALA 228:N,CA,C,O,CBPD498FINALMODEL:SER 229:N,CA,C,O,CB,OGPD498FINALMODEL:PRO 230:N,CA,CD,C,O,CB,CG
PD498FINALMODEL:HIS 231:N,CA,C,O,CB,
CG,ND1,CD2,CE1,NE2Subset RESTx:restxmole.listSubset RESTX:
NEWMODEL:233-234restxatom.listSubset RESTX:
NEWMODEL:ALA 233:N,CA,C,O,CB
NEWMODEL:GLY 234:N,CA,C,O实施例4:Arthromyces ramosus过氧化物酶中适于添加羧酸连接基团(-COOH)的合适取代
按照下述找出一种非水解酶-Arthromyces ramosus氧化物酶中适于添加羧基连接基团(天冬氨酸和谷氨酸)的合适位置。
在Brookhaven数据库中可以larp.pdb的文件名得到这种氧化还原酶的3D结构。此A.ramosus过氧化物酶中含有344个氨基酸残基。X-射线结构中见不到最初的8个残基:QGPGGGGG,N143被糖基化。
遵循实施例1中所述的操作。
Arthromyces ramosus过氧化物酶(E.C.1.11.1.7)的氨基酸序列示于SEQ ID NO:4中。
Insight(BIOSYM)中运行的各个指令示于下述指令文件makeDEzone.bcl和makeDEzone2.bcl中。C-末端残基是P344,ACTSITE定义为血红素基团和与其协作的两个组氨酸(H56和H184)。
保守性取代:makeDEzone.bclDelete Subset*Color Molecule Atoms * Specified Specification 255,0,255Zone Subset ASP:asp:od* Static monomer/residue 10 Color_Subset255,255,0Zone Subset GLU:glu:oe* Static monomer/residue 10 Color_Subset255,255,0#NOTE:editnextline C-terminal residue number according to theproteinZone Subset CTERM:344:O Static monomer/residue 10 Color_Subset255,255,0#NOTE:editnextline ACTSITE residues according to the proteinZone Subset ACTSITE:HEM,56,184 Static monomer/residue 8Color_Subset 255,255,0Combine Subset ALLZONE Union ASP GLUCombine Subset ALLZONE Union ALLZONE CTERMCombine Subset ALLZONE Union ALLZONE ACTSITE#NOTE:editnextline object name according to the proteinCombine Subset REST Difference ARP ALLZONEList Subset REST Atom Output_File restatom.listList Subset REST monomer/residue Output_File restmole.listColor Molecule Atoms ACTSITE Specified Specification 255,0,0List Subset ACTSITE Atom Output_File actsiteatom.listList Subset ACTSITE monomer/residue Output_Fileactsitemole.list#Zone Subset REST5A REST Static Monomer/Residue 5 -Color_SubsetCombine Subset SUB5A Difference REST5A ACTSITECombine Subset SUB5B Difference SUB5A RESTColor Molecule Atoms SUB5B Specified Specification 255,255,255List Subset SUB5B Atom Output_File sub5batom.listList Subset SUB5B monomer/residue Output_File sub5bmole.list#Now identify sites for asn→asp & gln→glu substitutions and...#continue with makezone2.bcl.#Use grep command to identify asn/gln in restatom.list ...#sub5batom.list & accsiteatom.list说明:
REST亚组包含Gln70,SUB5B包含Gln34,Asn128,Asn303,这几个残基都是溶剂暴露型的。
已鉴定出A.ramosus过氧化物酶内Q34E或Q34D、Q70E或Q70D、N128D或N128E以及N303D或N303E这些取代是适于进行诱变的一些位点。在下述将对这些残基进行取代,并进一步分析:
非保守性取代:makeDEzone2.bcl#sourcefile makezone2.bcl Claus von der Osten 961128##having scanned lists (grep gln/asn command) and identifiedsites for ...#asn→asp & gln→glu substitutions#NOTE:editnextline object name according to proteinCopy Object -To_Clipboard -Displace ARP newmodelBiopolymer#NOTE:editnextline object name according to proteinBlank Object On ARP#NOTE:editnextlines with asn→asp & gln→glu positionsReplace Residue newmodel:34 glu LReplace Residue newmodel:70 glu LReplace Residue newmodel:128 asp LReplace Residue newmodel:303 asp L##Now repeat analysis done prior to asn→asp & gln→glu,...#now including introduced asp & gluColor Molecule Atoms newmodel Specified Specification 255,0,255Zone Subset ASPx newmodel:asp:od* Static monomer/residue 10Color_Subset 255,255,0Zone Subset GLUx newmodel:glu:oe* Static monomer/residue 10Color_Subset 255,255,0#NOTE:editnextline C-terminal residue number according to theproteinZone Subset CTERMx newmodel:344:O Static monomer/residue 10Color_Subset 255,255,0#NOTE:editnextline ACTSITEx residues according to the proteinZone Subset ACTSITEx newmodel:HEM,56,184 Static monomer/residue8 Color_Subset 255,255,0Combine Subset ALLZONEx Union ASPx GLUxCombine Subset ALLZONEx Union ALLZONEx CTERMxCombine Subset ALLZONEx Union ALLZONEx ACTSITExCombine Subset RESTx Difference newmodel ALLZONExList Subset RESTx Atom Output_File restxatom.listList Subset RESTx monomer/residue Output_File restxmole.list#Color Molecule Atoms ACTSITEx Specified Specification 255,0,0List Subset ACTSITEx Atom Output_File actsitexatom.listList Subset ACTSITEx monomer/residue Output_Fileactsitexmole.list##read restxatom.list or restxmole.list to identify sites for(not_gluasp)→gluasp ...#subst. if needed说明:
RESTx亚组仅含有4个残基:S9、S334、G335和P336,它们的溶剂暴露程度都>5%。建议在A.ramosus过氧化物酶内进行下述突变:S9D、S9E、S334D、S334E、G335D、G335E、P336D和P336E。ACTSITE亚组内的酸性残基有:E44、D57、D77、E87、E176、D179、E190、D202、D209、D246以及P344上的N-末端羧酸。这些残基中仅E44、D77、E176、D179、E190、D209、D246以及P344上的N-末端羧酸是溶剂暴露型的。突变的合适位点是:E44Q、D77N、E176Q、D179N、E190Q、D209N和D246N。由于D246对血红素的结合比较重要,因此D246N和D246E这两种突变颇有危险。
由于N-末端8个残基不出现在此结构中,因此,在上述计算中不包括这几个残基。这8个残基,QGPGGGGG,都不含羧基。下述变体有可能使得能在这一区域内进行连接:Q1E、Q1D、G2E、G2D、P3E、P3D、G4E、G4D、G5E、G5D、G6E、G6D、G7E、G7D、G8E、G8D。
实施例4的有关数据:
A.ramosus氧化物酶的溶剂可及性数据(备注:由于X-射线结构中缺失了最开始的8个残基,因此,在下述可及性表中列出的残基号比其它处用于残基编号的残基号要小8)#ARP Thu Jan 30 15:39:05 MET 1997#残基 面积SER_1 143.698257VAL_2 54.879990THR_3 86.932701CYS_4 8.303715PRO_5 126.854782GLY_6 53.771488GLY_7 48.137802GLN_8 62.288475SER_9 79.932549THR_10 16.299215SER_11 81.928642ASN_12 51.432678SER_13 81.993019GLN_14 92.344009CYS_15 0.000000CYS_16 32.317432VAL_17 54.067810TRP_18 6.451035PHE_19 25.852070ASP_20 79.033997VAL_21 0.268693LEU_22 22.032858ASP_23 90.111404ASP_24 43.993240LEU_25 1.074774GLN_26 25.589321THR_27 82.698059ASN_28 96.600883PHE_29 32.375275TYR_30 5.898365GLN_31 103.380585GLY_32 40.042034SER_33 46.789322LYS_34 87.161873CYS_35 12.827215GLU_36 51.582657SER_37 16.378180PRO_38 33.560043VAL_39 6.448641ARG_40 7.068311LYS_41 15.291286ILE_42 1.612160LEU_43 1.880854ARG_44 16.906845ILE_45 0.000000VAL_46 2.312647PHE_47 2.955627HIS_48 20.392527ASP_49 4.238116ALA_50 0.510757ILE_51 1.576962GLY_52 2.858601PHE_53 48.633503SER_54 8.973248PRO_55 58.822315ALA_56 59.782852LEU_57 46.483955THR_58 86.744827ALA_59 89.515816ALA_60 81.163239GLY_61 70.119019GLN_62 112.635498PHE_63 93.522354GLY_64 2.742587GLY_65 13.379636GLY_66 22.722847GLY_67 0.000000ALA_68 0.268693ASP_69 12.074840GLY_70 0.700486SER_71 0.000000ILE_72 0.000000ILE_73 0.000000ALA_74 17.304443HIS_75 41.071186SER_76 20.000793ASN_77 120.855316ILE_78 66.574982GLU_79 2.334954LEU_80 41.329689ALA_81 77.370575PHE_82 38.758774PRO_83 131.946289ALA_84 34.893864ASN_85 5.457000GLY_86 43.364151GLY_87 51.561348LEU_88 0.242063THR_89 73.343575ASP_90 130.139389THR_91 17.863211ILE_92 0.268693GLU_93 92.210396ALA_94 35.445068LEU_95 1.343467ARG_96 31.175611ALA_97 44.650192VAL_98 17.698566GLY_99 1.471369ILE_100 62.441463ASN_101 107.139748HIS_102 46.952496GLY_103 46.559296VAL_104 11.342628SER_105 15.225677PHE_106 6.422011GLY_107 3.426864ASP_108 10.740790LEU_109 0.268693ILE_110 1.880854GLN_111 31.867456PHE_112 0.000000ALA_113 0.000000THR_114 3.656114ALA_115 8.299393VAL_116 0.268693GLY_117 0.268693MET_118 3.761708SER_119 14.536770ASN_120 25.928799CYS_121 0.537387PRO_122 29.798336GLY_123 33.080013SER_124 17.115562PRO_125 36.908714ARG_126 108.274727LEU_127 21.238588GLU_128 53.742313PHE_129 3.761708LEU_130 12.928699THR_131 10.414591GLY_132 47.266495ARG_133 12.247048SER_134 63.047237ASN_135 31.403708SER_136 97.999619SER_137 28.505201GLN_138 102.845520PRO_139 49.691917SER_140 9.423104PRO_141 25.724171PRO_142 80.706665SER_143 105.318176LEU_144 20.154398ILE_145 41.288322PRO_146 10.462679GLY_147 19.803421PRO_148 18.130360GLY_149 47.391853ASN_150 60.248917THR_151 87.887985VAL_152 13.870322THR_153 74.664734ALA_154 45.251106ILE_155 2.686934LEU_156 28.720940ASP_157 110.081253ARG_158 31.228874MET_159 1.612160GLY_160 38.223858ASP_161 46.293152ALA_162 9.877204GLY_163 34.267326PHE_164 11.057570SER_165 51.158882PRO_166 62.767738ASP_167 75.164917GLU_168 43.334976VAL_169 6.365355VAL_170 2.955627ASP_171 7.004863LEU_172 1.880854LEU_173 3.197691ALA_174 0.000000ALA_175 1.074774HIS_176 0.502189SER_177 0.806080LEU_178 3.197691ALA_179 3.337480SER_180 0.466991GLN_181 2.122917GLU_182 40.996552GLY_183 62.098671LEU_184 23.954853ASN_185 15.918136SER_186 95.185318ALA_187 59.075272ILE_188 27.675419PHE_189 102.799423ARG_190 55.265549SER_191 6.986028PRO_192 2.686934LEU_193 12.321225ASP_194 2.127163SER_195 33.556419THR_196 33.049286PRO_197 20.874798GLN_198 65.729698VAL_199 31.705818PHE_200 4.753195ASP_201 13.744506THR_202 1.612160GLN_203 16.081930PHE_204 2.581340TYR_205 1.880854ILE_206 9.356181GLU_207 0.735684THR_208 10.685907LEU_209 9.672962LEU_210 2.955627LYS_211 77.176834GLY_212 40.968609THR_213 78.718216THR_214 21.738384GLN_215 77.622299PRO_216 25.441587GLY_217 8.320850PRO_218 96.972305SER_219 64.627823LEU_220 85.732414GLY_221 27.361111PHE_222 134.620178ALA_223 3.873014GLU_224 12.141763GLU_225 65.129868LEU_226 76.105843SER_227 0.268693PRO_228 7.017754PHE_229 0.000000PRO_230 47.827423GLY_231 23.790522GLU_232 6.643466PHE_233 6.713862ARG_234 18.012030MET_235 4.598188ARG_236 91.415581SER_237 1.982125ASP_238 6.246871ALA_239 12.897283LEU_240 76.820526LEU_241 3.224321ALA_242 1.400973ARG_243 77.207176ASP_244 36.207306SER_245 104.023796ARG_246 121.852341THR_247 2.955627ALA_248 4.810700CYS_249 47.331306ARG_250 62.062778TRP_251 2.418241GLN_252 5.554953SER_253 38.284832MET_254 1.124224THR_255 0.000000SER_256 53.758987SER_257 37.276134ASN_258 44.381340GLU_259 149.565140VAL_260 57.500389MET_261 2.679314GLY_262 10.175152GLN_263 107.458916ARG_264 36.402130TYR_265 0.233495ARG_266 91.179619ALA_267 53.708500ALA_268 6.504294MET_269 17.122011ALA_270 22.455158LYS_271 73.386177MET_272 3.959508SER_273 15.043281VAL_274 23.887930LEU_275 17.196379GLY_276 44.362202PHE_277 68.062485ASP_278 94.902039ARG_279 113.549011ASN_280 134.886017ALA_281 72.340973LEU_282 26.692348THR_283 27.696728ASP_284 72.214157CYS_285 0.000000SER_286 28.209335ASP_287 64.560753VAL_288 7.040061ILE_289 8.665112PRO_290 48.682365SER_291 86.141670ALA_292 29.031240VAL_293 84.432014SER_294 85.944153ASN_295 49.017288ASN_296 133.459198ALA_297 57.283794ALA_298 65.233749PRO_299 24.751518VAL_300 45.409184ILE_301 8.060802PRO_302 14.742939GLY_303 16.589832GLY_304 34.238071LEU_305 24.719791THR_306 49.356300VAL_307 71.491821ASP_308 130.906174ASP_309 31.733070ILE_310 19.581894GLU_311 81.414574VAL_312 94.769890SER_313 39.688896CYS_314 9.998511PRO_315 120.328018SER_316 95.364319GLU_317 65.560959PRO_318 100.254364PHE_319 46.284115PRO_320 31.328060GLU_321 177.602249ILE_322 33.449741ALA_323 46.892982THR_324 79.976471ALA_325 36.423820SER_326 124.467422GLY_327 28.219524PRO_328 107.553696LEU_329 86.789825PRO_330 34.287163SER_331 75.764053LEU_332 32.840569ALA_333 61.516434PRO_334 82.389992ALA_335 6.246871PRO_336 56.750813HEM_337 60.435017CA_338 2.078997CA_339 0.000000NAG_340 141.534668NAG_341 186.311371Subset REST:restmole.listSubset REST:
ARP:9,69-70,125,127,133,299-301,334-336restatom.listSubset REST:
ARP:SER 9:N,CA,C,O,CB,OG
ARP:GLY 69:N,CA,C,O
ARP:GLN 70:N,CA,C,O,CB,CG,CD,OE1,NE2
ARP:GLY 125:N,CA,C,O
ARP:SER 127:N,CA,C,O,CB,OG
ARP:PRO 133:N,CA,CD,C,O,CB,CG
ARP:SER 299:N,CA,C,O,CB,OG
ARP:ALA 300:N,CA,C,O,CB
ARP:VAL 301:N,CA,C,O,CB,CG1,CG2
ARP:SER 334:N,CA,C,O,CB,OG
ARP:GLY 335:N,CA,C,O
ARP:PRO 336:N,CA,CD,C,O,CB,CGSubset SUB5B:sub5bmole.listSubset SUB5B:
ARP:10-11,34,38,65-68,71-72,120-121,123-124,
128-132,134,270,274,
ARP:297-298,302-303,311-312,332-333,337-338sub5batom.listSubset SUB5B:
ARP:VAL 10:N,CA,C,O,CB,CG1,CG2
ARP:THR 11:N,CA,C,O,CB,OG1,CG2
ARP:GLN 34:N,CA,C,O,CB,CG,CD,OE1,NE2
ARP:TYR 38:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH
ARP:LEU 65:N,CA,C,O,CB,CG,CD1,CD2
ARP:THR 66:N,CA,C,O,CB,OG1,CG2
ARP:ALA 67:N,CA,C,O,CB
ARP:ALA 68:N,CA,C,O,CB
ARP:PHE 71:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ
ARP:GLY 72:N,CA,C,O
ARP:PHE 120:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ
ARP:ALA 121:N,CA,C,O,CB
ARP:ALA 123:N,CA,C,O,CB
ARP:VAL 124:N,CA,C,O,CB,CG1,CG2
ARP:ASN 128:N,CA,C,O,CB,CG,OD1,ND2
ARP:CYS 129:N,CA,C,O,CB,SG
ARP:PRO 130:N,CA,CD,C,O,CB,CG
ARP:GLY 131:N,CA,C,O
ARP:SER 132:N,CA,C,O,CB,OG
ARP:ARG 134:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2
ARP:GLY 270:N,CA,C,O
ARP:ARG 274:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2
ARP:ILE 297:N,CA,C,O,CB,CG1,CG2,CD1
ARP:PRO 298:N,CA,CD,C,O,CB,CG
ARP:SER 302:N,CA,C,O,CB,OG
ARP:ASN 303:N,CA,C,O,CB,CG,OD1,ND2
ARP:GLY 311:N,CA,C,O
ARP:GLY 312:N,CA,C,O
ARP:THR 332:N,CA,C,O,CB,OG1,CG2
ARP:ALA 333:N,CA,C,O,CB
ARP:LEU 337:N,CA,C,O,CB,CG,CD1,CD2
ARP:PRO 338:N,CA,CD,C,O,CB,CGSubset ACTSITE:actsitemole.listSubset ACTSITE:
ARP:44-61,75-77,79-80,87-88,90-96,
99,118,122,126,135,148-149,152-158,
ARP:163-164,167,176-194,197-205,207-209,211-
213,216,230-231,241,
ARP:243-246,249,259,273,277,280,343-347Hactsiteatom.listSubset ACTSITE:
ARP:GLU 44:N,CA,C,O,CB,CG,CD,OE1,OE2
ARP:SER 45:N,CA,C,O,CB,OG
ARP:PRO 46:N,CA,CD,C,O,CB,CG
ARP:VAL 47:N,CA,C,O,CB,CG1,CG2
ARP:ARG 48:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2
ARP:LYS 49:N,CA,C,O,CB,CG,CD,CE,NZ
ARP:ILE 50:N,CA,C,O,CB,CG1,CG2,CD1
ARP:LEU 51:N,CA,C,O,CB,CG,CD1,CD2
ARP:ARG 52:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2
ARP:ILE 53:N,CA,C,O,CB,CG1,CG2,CD1
ARP:VAL 54:N,CA,C,O,CB,CG1,CG2
ARP:PHE 55:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ
ARP:HIS 56:N,CA,C,O,CB,CG,ND1,CD2,CE1,NE2
ARP:ASP 57:N,CA,C,O,CB,CG,OD1,OD2
ARP:ALA 58:N,CA,C,O,CB
ARP:ILE 59:N,CA,C,O,CB,CG1,CG2,CD1
ARP:GLY 60:N,CA,C,O
ARP:PHE 61:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ
ARP:GLY 75:N,CA,C,O
ARP:ALA 76:N,CA,C,O,CB
ARP:ASP 77:N,CA,C,O,CB,CG,OD1,OD2
ARP:SER 79:N,CA,C,O,CB,OG
ARP:ILE 80:N,CA,C,O,CB,CG1,CG2,CD1
ARP:GLU 87:N,CA,C,O,CB,CG,CD,OE1,OE2
ARP:LEU 88:N,CA,C,O,CB,CG,CD1,CD2
ARP:PHE 90:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ
ARP:PRO 91:N,CA,CD,C,O,CB,CG
ARP:ALA 92:N,CA,C,O,CB
ARP:ASN 93:N,CA,C,O,CB,CG,OD1,ND2
ARP:GLY 94:N,CA,C,O
ARP:GLY 95:N,CA,C,O
ARP:LEU 96:N,CA,C,O,CB,CG,CD1,CD2
ARP:THR 99:N,CA,C,O,CB,OG1,CG2
ARP:ILE 118:N,CA,C,O,CB,CG1,CG2,CD1
ARP:THR 122:N,CA,C,O,CB,OG1,CG2
ARP:MET 126:N,CA,C,O,CB,CG,SD,CE
ARP:LEU 135:N,CA,C,O,CB,CG,CD1,CD2
ARP:SER 148:N,CA,C,O,CB,OG
ARP:PRO 149:N,CA,CD,C,O,CB,CGARP:LEU 152:N,CA,C,O,CB,CG,CD1,CD2ARP:ILE 153:N,CA,C,O,CB,CG1,CG2,CD1ARP:PRO 154:N,CA,CD,C,O,CB,CGARP:GLY 155:N,CA,C,OARP:PRO 156:N,CA,CD,C,O,CB,CGARP:GLY 157:N,CA,C,OARP:ASN 158:N,CA,C,O,CB,CG,OD1,ND2ARP:ILE 163:N,CA,C,O,CB,CG1,CG2,CD1ARP:LEU 164:N,CA,C,O,CB,CG,CD1,CD2ARP:MET 167:N,CA,C,O,CB,CG,SD,CEARP:GLU 176:N,CA,C,O,CB,CG,CD,OE1,OE2ARP:VAL 177:N,CA,C,O,CB,CG1,CG2ARP:VAL 178:N,CA,C,O,CB,CG1,CG2ARP:ASP 179:N,CA,C,O,CB,CG,OD1,OD2ARP:LEU 180:N,CA,C,O,CB,CG,CD1,CD2ARP:LEU 181:N,CA,C,O,CB,CG,CD1,CD2ARP:ALA 182:N,CA,C,O,CBARP:ALA 183:N,CA,C,O,CBARP:HIS 184:N,CA,C,O,CB,CG,ND1,CD2,CE1,NE2ARP:SER 185:N,CA,C,O,CB,OGARP:LEU 186:N,CA,C,O,CB,CG,CD1,CD2ARP:ALA 187:N,CA,C,O,CBARP:SER 188:N,CA,C,O,CB,OGARP:GLN 189:N,CA,C,O,CB,CG,CD,OE1,NE2ARP:GLU 190:N,CA,C,O,CB,CG,CD,OE1,OE2ARP:GLY 191:N,CA,C,OARP:LEU 192:N,CA,C,O,CB,CG,CD1,CD2ARP:ASN 193:N,CA,C,O,CB,CG,OD1,ND2ARP:SER 194:N,CA,C,O,CB,OGARP:PHE 197:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZARP:ARG 198:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2ARP:SER 199:N,CA,C,O,CB,OGARP:PRO 200:N,CA,CD,C,O,CB,CGARP:LEU 201:N,CA,C,O,CB,CG,CD1,CD2ARP:ASP 202:N,CA,C,O,CB,CG,OD1,OD2ARP:SER 203:N,CA,C,O,CB,OGARP:THR 204:N,CA,C,O,CB,OG1,CG2ARP:PRO 205:N,CA,CD,C,O,CB,CGARP:VAL 207:N,CA,C,O,CB,CG1,CG2ARP:PHE 208:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZARP:ASP 209:N,CA,C,O,CB,CG,OD1,OD2ARP:GLN 211:N,CA,C,O,CB,CG,CD,OE1,NE2ARP:PHE 212:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZARP:TYR 213:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OHARP:THR 216:N,CA,C,O,CB,OG1,CG2ARP:PHE 230:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZARP:ALA 231:N,CA,C,O,CBARP:PHE 241:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZARP:MET 243:N,CA,C,O,CB,CG,SD,CEARP:ARG 244:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2ARP:SER 245:N,CA,C,O,CB,OGARP:ASP 246:N,CA,C,O,CB,CG,OD1,OD2ARP:LEU 249:N,CA,C,O,CB,CG,CD1,CD2ARP:TRP 259:N,CA,C,O,CB,CG,CD1,
CD2,NE1,CE2,CE3,CZ2,CZ3,CH2ARP:TYR 273:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OHARP:MET 277:N,CA,C,O,CB,CG,SD,CE
ARP:MET 280:N,CA,C,O,CB,CG,SD,CE
ARP:ALA 343:N,CA,C,O,CB
ARP:PRO 344:N,CA,CD,C,O,OXT,CB,CG
ARP:HEM 345H:FE,NA,NB,NC,ND,CHA,CHB,
CHC,CHD,C1A,C2A,C3A,CMA,CMA,CAA,CBA,CGA
ARP:HEM 345H:O1A,O2A,C1B,C2B,C3B,C4B,CMB,
CAB,CBB,C1C,C2C,C3C,C4C,CMC,CAC,CBC
ARP:HEM 345H:C1D,C2D,C3D,C4D,CMD,CAD,CBD,CGD,O1D,O2D
ARP:CA 346H:CA
ARP:CA 347H:CASubset RESTx:restxmole.listSubset RESTX
NEWMODEL:9,334-336restxatom.listSubset RESTX:
NEWMODEL:SER 9:N,CA,C,O,CB,OG
NEWMODEL:SER 334:N,CA,C,O,CB,OG
NEWMODEL:GLY 335:N,CA,C,O
NEWMODEL:PRO 336:N,CA,CD,C,O,CB,CG实施例5:用N-琥珀酰亚胺碳酸酯活化mPEG 15,000
将mPEG 15,000悬浮在甲苯中(4ml/g mPEG),在正常压力下蒸馏除去20%以共沸干燥反应物。当溶液冷却到30℃时,加入二氯甲烷(1ml/g干燥mPEG),并加入甲苯中的碳酰氯(1.93M 5摩尔/摩尔mPEG),混合物在室温下搅动过夜。混合物蒸发至干燥,所得的目的产物为蜡状块。
在蒸发之后,添加二氯甲烷和甲苯(1∶2,3ml/g干燥mPEG)以重新溶解白色固体。以固体形式加入N-羟基琥珀酰亚胺(2摩尔/摩尔mPEG),然后加入三乙胺(1.1摩尔/摩尔mPEG)。将混合物搅动3小时。混合物最初并不清亮,然后变得清亮,最后形成小沉淀。将混合物蒸发至干燥,在乙酸乙酯(10ml)中重结晶,热过滤以除去盐和不溶性的微量物质。放置清亮液体,使其在室温下缓慢冷却16小时,然后在冰箱中过夜。滤出白色沉淀,以一些冷乙酸乙酯洗涤,并进行干燥,得到98%(w/w)的产物。核磁共振表明有80-90%被活化和5o/oo(w/w)HNEt3Cl。mPEG 15,000(CDCl3)的1H-NMR为:d 1.42t(I=4.8CH3i HNEt3Cl),2.84s(I=3.7琥珀酰亚胺),3.10dq(I=3.4 CH2i HNEt3Cl),3.38s(I=2.7 CH3 i OMe),3.40dd(I=4.5o/oo13C satellite),3.64 bs(I=1364主峰),3.89 dd(I=4.8o/oo13C satellite),4.47dd(I=1.8,PEG中的CH2)。于22℃下在干燥器中存储4个月后来见变化。实施例6:用N-琥珀酰亚胺碳酸酯活化mPEG 5,000
如实施例5所述,用N-琥珀酰亚胺碳酸酯活化mPEG 5,000。实施例7:PD498变体的构建和表达
采用Sarkar等人(1990,生物技术,8:404-407)描述的“maxi-寡核苷酸PCR”方法,构建PD498定点变体。
模板质粒是含有PD498蛋白酶基因变体的穿梭载体pPD498或者其类似物。
构建、表达和纯化下述PD498变体。A:R28KB:R62KC:R169KD:R28K+R62KE:R28K+R169KF:R62K+R169KG:R28K+R69K+R169K构建变体
为了导入R28K取代,利用了含有下述序列的一种合成寡核苷酸:GGG ATG TAA CCA AGG GAA GCA GCA CTC AAA CG(SEQ ID NO:7)。
将769bp的PCR片段连接到经过BstE II和BglII消化的pPD498质粒中。通过StyI消化检测出阳性变体,并通过对此整个769bp插入片段的DNA测序进一步证实。
为了导入R62K取代,利用了含有下述序列的一种合成寡核苷酸:CGA CTT TAT CGA TAA GGA CAA TAA CCC(SEQ ID NO:8)。
将769bp的PCR片段连接到经过BstE II和BglII消化的pPD498质粒中。通过ClaI消化检测出阳性变体,并通过对此整个769bp插入片段的DNA测序进一步证实。
为了导入R169K取代,利用了含有下述序列的一种合成寡核苷酸:CAA TGT ATC CAA AAC GTT CCA ACC AGC(SEQ ID NO:9)。
将769bp的PCR片段连接到经过BstE II和BglII消化的pPD498质粒中。通过一个RsaI限制性位点的缺失检测出阳性变体,并通过对此整个769bp插入片段的DNA测序进一步证实。
为了同时导入R28K和R62K取代,同时利用了含有下述序列的合成寡核苷酸:GGG ATG TAA CCA AGG GAA GCA CCA CTC AAA CG(SEQ ID NO:7)以及CGA CTT TAT CGA TAA GGA CAA TAA CCC(SEQ ID NO:8)。将769bp的PCR片段连接到经过BstE II和BglII消化的pPD498质粒中。通过StyI和ClaI消化检测出阳性变体,并通过对此整个769bp插入片段的DNA测序进一步证实。
为了同时导入R28K和R169K取代,同时利用了含有下述序列的合成寡核苷酸:GGG ATG TAA CCA AGG GAA GCA GCA CTC AAA CG(SEQ ID NO:8)以及CAA TGT ATC CAA AAC GTT CCA ACC AGC(SEQ ID NO:8)。将769bp的PCR片段连接到经过BstE II和BglII消化的pPD498质粒中。通过StyI的消化以及一个RsaI位点的缺失检测出阳性变体。通过对此整个769bp插入片段的DNA测序进一步证实。
为了同时导入R62K和R169K取代,同时利用了含有下述序列的合成寡核苷酸:CGA CTT TAT CGA TAA GGA CAA TAA CCC(SEQ ID NO:8),以及CAA TGT ATC CAA AAC GTT CCA ACC AGC(SEQ ID NO:9)。将769bp的PCR片段连接到经过BstEII和BglII消化的pPD498质粒中。通过ClaI的消化以及一个RsaI位点的缺失检测出阳性变体。通过对此整个769bp插入片段的DNA测序进一步证实。
为了同时导入R28K、R62K和R169K取代,同时利用了分别含有下述序列的几种合成寡核苷酸:GGG ATG TAA CCA AGG GAA GCA GCA CTC AAA CG(SEQ ID NO:7)CGA CTT TAT CGA TAA GGA CAA TAA CCC(SEQ ID NO:8),以及CAA TGT ATC CAA AAC GTT CCA ACC AGC(SEQ ID NO:9)。将769bp的PCR片段连接到经过BstEII和BglII消化的pPD498质粒中。通过StyI和ClaI的消化以及一个RsaI位点的缺失检测出阳性变体。通过对此整个769bp插入片段的DNA测序进一步证实。PD498变体的发酵、表达和纯化
从大肠杆菌培养物中纯化出含有上述PD498变体的载体,将它们转化至枯草芽孢杆菌中,按照上述“材料和方法”所述,从中发酵、表达和纯化变体。实施例7:三联体取代的PD498变体与活化的mPEG 5,000的偶联
在20ml的终体积内,于50mM硼酸钠(pH 10)中,将200mg三联体取代的PD498变体(即R28K+R62K+R169K取代的变体)与1.8g用N-琥珀酰亚胺碳酸酯活化的mPEG 5,000(按照实施例2制备)一起温育。通过磁性搅拌,在室温下进行反应。反应时间是1小时。通过添加DMG缓冲液至终浓度为5mM二甲基戊二酸、1mM CaCl2和50mM硼酸盐(pH 5.0)来终止反应。
所得衍生物的分子量大约是120kDa,相当于每摩尔酶连接了大约16摩尔mPEG。
与亲本酶比较,被修饰酶对肽底物(琥珀酰-Ala-Ala-Pro-Phe-酰基对硝基苯胺)的剩余活性接近100%。实施例8:豚鼠中PD498变体-SPEG 5,000的变应原性实验
通过气管内施药,用1.0μg PD498-SPEG 5,000和1.0μg修饰的变体PD498-SPEG 5,000刺激Dunkin Hartley豚鼠。
用特异性的IgG1 ELISA(如上所述)检测实验期间经免疫的DunkinHartley豚鼠的血清,以分析该分子是否能活化免疫应答系统,引起指示为变应原性应答的特异性IgG1应答。
观察10周的实验期间Dunkin Hartley豚鼠的IgG1水平。实施例9:Humicola lanuginosa脂肪酶中适于添加氨基连接基团(-NH2)的取代
在Brookhaven数据库中,可以1tib.pdb的文件名得到Humicolalanuginosa脂肪酶(SEQ ID NO:6)的3D结构。该脂肪酶由269个氨基酸组成。
遵循实施例1中描述的操作。在下面列出的H.lanuginosa脂肪酶溶剂可及性数据中给出了H.lanuginosa脂肪酶的序列。采用H.lanuginosa残基编号系统(1-269),活性位点残基(功能性位点)是S146、S201和H258。用同义词TIB表示H.lanuginosa脂肪酶。
Insight(BIOSYM)中运行的各个指令示于下述指令文件makeKzone.bcl和makeKzone2.bcl中:
保守性取代:makeKzone.bcl1 Delete Subset *2 Color Molecule Atoms * Specified Specification 255,0,2553 Zone Subset LYS:lys:NZ Static monomer/residue 10Color_Subset 255,255,04 Zone Subset NTERM:1:N Static monomer/residue 10Color_Subset 255,255,05 #NOTE:editnextline ACTSITE residues according to theprotein6 Zone Subset ACTSITE:146,201,258 Static monomer/residue 8Color_Subset 255,255,07 Combine Subset ALLZONE Union LYS NTERM8 Combine Subset ALLZONE Union ALLZONE ACTSITE9 #NOTE:editnextline object name according to the protein10 Combine Subset REST Difference TIB ALLZONE11 List Subset REST Atom Output_File restatom.list12 List Subset REST monomer/residue Output_File restmole.list13 Color Molecule Atoms ACTSITE Specified Specification 255,0,014 List Subset ACTSITE Atom Output_File actsiteatom.list15 List Subset ACTSITE monomer/residue Output_Fileactsitemole.list16 #17 Zone Subset REST5A REST Static Monomer/Residue 5 -Color_Subset18 Combine Subset SUB5A Difference REST5A ACTSITE19 Combine Subset SUB5B Difference SUB5A REST20 Color Molecule Atoms SUB5B Specified Specification255,255,25521 List Subset SUB5B Atom Output_File sub5batom.list22 List Subset SUB5B monomer/residue Output_File sub5bmole.list23 #Now identify sites for lys→arg substitutions and continuewith makezone2.bcl24 #Use grep command to identify ARG in restatom.list,sub5batom.list & accsiteatom.list说明:
在H.lanuginosa脂肪酶(=TIB)的情形下,REST包含精氨酸Arg133、Arg139、Arg160、Arg179和Arg209,SUB5B包含Arg118和R125。
这些残基均是溶剂暴露型的。已鉴定出TIB内R133K,R139K,R160K,R179K,R209K,R118K和R125K这些取代是适于进行本发明范围内诱变的一些位点。在下述第2部分将对这些残基进行取代,并进一步分析。ACTSITE亚组中无赖氨酸。
非保守性取代:makeKzone2.bcl1 #sourcefile makezone2.bcl Claus von der Osten 9611282 #3 #having scanned lists (grep arg command) and identifiedsites for lys→arg substitutions4 #NOTE:editnextline object name according to protein5 Copy Object -To_Clipboard -Displace TIB newmodel6 Biopolymer7 #NOTE:editnextline object name according to protein8 Blank Object On TIB9 #NOTE:editnextlines with lys→arg positions10 Replace Residue newmodel:118 lys L11 Replace Residue newmodel:125 lys L12 Replace Residue newmodel:133 lys L13 Replace Residue newmodel:139 lys L14 RePlace Residue newmodel:160 lys L15 Replace Residue newmodel:179 lys L16 Replace Residue newmodel:209 lys L17 #18 #Now repeat analysis done prior to arg→lys,now includingintroduced lysines19 Color Molecule Atoms newmodel Specified Specification255,0,25520 Zone Subset LYSx newmodel:lys:NZ Static monomer/residue 10Color_Subset 255,255,021 Zone Subset NTERMx newmodel:1:N Static monomer/residue 10Color_Subset 255,255,022 #ZOTE:editnextline ACTSITEx residues according to theprotein23 Zone Subset ACTSITEx newmodel:146,201,258 Staticmonomer/residue 8 Color_Subset 255,255,024 Combine Subset ALLZONEx Union LYSx NTERMx25 Combine Subset ALLZONEx Union ALLZONEx ACTSITEx26 Combine Subset RESTx Difference newmodel ALLZONEx27 List Subset RESTx Atom Output File_restxatom.list28 List Subset RESTx monomer/residue Output_Filerestxmole.list29 #30 Color Molecule Atoms ACTSITEx Specified Specification255,0,031 List Subset ACTSITEx Atom Output_File actsitexatom.list32 List Subset ACTSITEx monomer/residue Output_Fileactsitexmole.list33 #34 #read restxatom.list or restxmole.list to identify sitesfor (not_arg)→lys subst.if needed说明:
在RESTx亚组内的残基中,下述残基的暴露程度>5%(参见下表):18、31-33、36、38、40、48、50、56-62、64、78、88、91-93、104-106、120、136、225、227-229、250、262、268。这些残基中,有3个半胱氨酸参与二硫键形成,因而由于结构上的原因,对它们不进行突变。建议在H.lanuginosa脂肪酶(TIB)中进行下述突变:A18K,G31K,T32K,N33K,G38K,A40K,D48K,T50K,E56K,D57K,S58K,G59K,V60K,G61K,D62K,T64K,L78K,N88K,G91K,N92K,L93K,S105K,G106K,V120K,P136K,G225K,L227K,V228K,P229K,P250K,F262K。实施例2的有关数据:# TIBNOH2O#残基 面积GLU_1 110.792610VAL_2 18.002457SER_3 53.019516GLN_4 85.770164ASP_5 107.565826LEU_6 33.022659PHE_7 34.392754ASN_8 84.855331GLN_9 39.175591PHE_10 2.149547ASN_11 40.544380LEU_12 27.648788PHE_13 2.418241ALA_14 4.625293GLN_15 28.202387TYR_16 0.969180SER_17 0.000000ALA_18 7.008336ALA_19 0.000000ALA_20 0.000000TYR_21 6.947358CYS_22 8.060802GLY_23 32.147034LYS_24 168.890747ASN_25 8.014721ASN_26 11.815564ASP_27 92.263428ALA_28 18.206699PRO_29 83.188431ALA_30 69.428421GLY_31 50.693439THR_32 52.171135ASN_33 111.230743ILE_34 2.801945THR_35 82.130569CYS_36 17.269245THR_37 96.731941GLY_38 77.870995ASN_39 123.051003ALA_40 27.985256CYS_41 0.752820PRO_42 46.258949GLU_43 69.773987VAL_44 0.735684GLU_45 77.169510LYS_46 141.213562ALA_47 10.249716ASP_48 109.913902ALA_49 2.602721THR_50 32. 012184PHE_51 8.255627LEU_52 60.093613TYR_53 77.877937SER_54 26.980494PHE_55 10.747735GLU_56 112.689758ASP_57 92.064278SER_58 32.990780GLY_59 53.371807VAL_60 83.563644GLY_61 69.625633ASP_62 75.520988VAL_63 4.030401THR_64 8.652839GLY_65 0.000000PHE_66 0.268693LEU_67 11.822510ALA_68 0.537387LEU_69 30.243870ASP_70 0.000000ASN_71 84.101044THR_72 89.271126ASN_73 70.742401LYS_74 98.319168LEU_75 8.329495ILE_76 5.197878VAL_77 0.806080LEU_78 5.293978SER_79 0.000000PHE_80 2.079151ARG_81 41.085312GLY_82 1.471369SER_83 43.794014ARG_84 100.261627SER_85 70.607552ILE_86 59.696865GLU_87 136.510773ASN_88 119.376373TRP_89 102.851227ILE_90 78.068588GLY_91 60.783607ASN_92 45.769428LEU_93 134.228363ASN_94 101.810959PHE_95 41.212212ASP_96 79.645950LEU_97 25.281572LYS_98 88.840263GLU_99 132.377090ILE_100 9.135575ASN_101 63.444527ASP_102 88.652847ILE_103 33.470661CYS_104 11.553816SER_105 99.461174GLY_106 40.325161CYS_107 4.433561ARG_108 97.450104GLY_109 1.343467HIS_110 4.652464ASP_111 37.023655GLY_112 29.930408PHE_113 14.976435THR_114 10.430954SER_115 40.606895SER_116 13.462922TRP_117 10.747735ARG_118 114.364281SER_119 46.880249VAL_120 13.434669ALA_121 18.258261ASP_122 110.753098THR_123 69.641922LEU_124 17.090784ARG_125 73.929977GLN_126 101.320190LYS_127 84.450241VAL_128 6.448641GLU_129 47.700993ASP_130 75.529091ALA_131 11.340775VAL_132 27.896025ARG_133 153.136490GLU_134 132.140594HIS_135 54.553406PRO_136 97.386963ASP_137 22.653191TYR_138 35.392658ARG_139 74.321243VAL_140 10.173222VAL_141 0.233495PHE_142 3.224321THR_143 0.000000GLY_144 0.000000HIS_145 4.514527SER_146 15.749787LEU_147 40.709171GLY_148 0.000000GLY_149 0.000000ALA_150 0.537387LEU_151 22.838938ALA_152 0.268693THR_153 18.078798VAL_154 7.254722ALA_155 0.000000GLY_156 0.000000ALA_157 15.140230ASP_158 41.645477LEU_159 6.144750ARG_160 41.939716GLY_161 68.978180ASN_162 68.243805GLY_163 79.181274TYR_164 36.190247ASP_165 103.068283ILE_166 0.000000ASP_167 24.326443VAL_168 4.299094PHE_169 0.466991SER_170 3.339332TYR_171 0.000000GLY_172 0.000000ALA_173 12.674671PRO_174 13.117888ARG_175 10.004488VAL_176 21.422220GLY_177 2.680759ASN_178 21.018063ARG_179 110.282166ALA_180 33.210381PHE_181 4.567788ALA_182 3.897251GLU_183 76.354004PHE_184 71.225983LEU_185 24.985012THR_186 47.023815VAL_187 98.244606GLN_188 54.152954THR_189 88.660645GLY_190 24.792120GLY_191 10.726818THR_192 45.458744LEU_193 16.633211TYR_194 34.829491ARG_195 29.030851ILE_196 1.973557THR_197 3.493014HIS_198 1.532270THR_199 34.785877ASN_200 39.789238ASP_201 0.000000ILE_202 31.168434VAL_203 29.521076PRO_204 3.515322ARG_205 44.882454LEU_206 51.051746PRO_207 12.575329PRO_208 43.259636ARG_209 113.700233GLU_210 154.628540PHE_211 112.505188GLY_212 30.084938TYR_213 3.268936SER_214 12.471436HIS_215 23.354481SER_216 16.406200SER_217 14.665598PRO_218 17.240993GLU_219 13.145291TYR_220 18.718306TRP_221 39.229233ILE_222 5.105175LYS_223 120.739983SER_224 15.407301GLY_225 29.306646THR_226 66.806862LEU_227 122.682808VAL_228 60.923004PRO_229 104.620377VAL_230 23.398251THR_231 63.372971ARG_232 80.357857ASN_233 89.255066ASP_234 43.011250ILE_235 2.114349VAL_236 45.140491LYS_237 105.651306ILE_238 24.671705GLU_239 116.891907GLY_240 31.965794ILE_241 46.278099ASP_242 28.963699ALA_243 25.158146THR_244 98.351440GLY_245 43.842186GLY_246 0.700486ASN_247 3.926274ASN_248 51.047890GLN_249 66.699188PRO_250 132.414047ASN_251 70.213730ILE_252 141.498062PRO_253 59.089233ASP_254 59.010895ILE_255 63.298943PRO_256 78.608688ALA_257 0.806080HIS_258 3.761708LEU_259 50.747856TRP_260 35.229710TYR_261 5.440791PHE_262 36.457939GLY_263 22.071375LEU_264 109.148178ILE_265 2.418241GLY_266 17.730062THR_267 68.217873CYS_268 15.418195LEU_269 165.990997Subset REST:restmole.listSubset REST:
TIB:5,8-9,13-14,16,18-20,31-34,36,38,40,48-50,56-
66,68,76-79,88,91-93,
TIB:100-107,116-117,119-121,132-134,136,139-142,154-169,177-185,
TIB:187,189-191,207-212,214-216,225,227-229,241-
244,250,262,268restatom. listSubset REST:
TIB:ASP 5:N,CA,C,O,CB,CG,OD1,OD2
TIB:ASN 8:N,CA,C,O,CB,CG,OD1,ND2
TIB:GLN 9:N,CA,C,O,CB,CG,CD,OE1,NE2
TIB:PHE 13:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ
TIB:ALA 14:N,CA,C,O,CB
TIB:TYR 16:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH
TIB:ALA 18:N,CA,C,O,CB
TIB:ALA 19:N,CA,C,O,CB
TIB:ALA 20:N,CA,C,O,CB
TIB:GLY 31:N,CA,C,O
TIB:THR 32:N,CA,C,O,CB,OG1,CG2
TIB:ASN 33:N,CA,C,O,CB,CG,OD1,ND2
TIB:ILE 34:N,CA,C,O,CB,CG1,CG2,CD1TIB:CYS 36:N,CA,C,O,CB,SGTIB:GLY 38:N,CA,C,OTIB:ALA 40:N,CA,C,O,CBTIB:ASP 48:N,CA,C,O,CB,CG,OD1,OD2TIB:ALA 49:N,CA,C,O,CBTIB:THR 50:N,CA,C,O,CB,OG1,CG2TIB:GLU 56:N,CA,C,O,CB,CG,CD,OE1,OE2TIB:ASP 57:N,CA,C,O,CB,CG,OD1,OD2TIB:SER 58:N,CA,C,O,CB,OGTIB:GLY 59:N,CA,C,OTIB:VAL 60:N,CA,C,O,CB,CG1,CG2TIB:GLY 61:N,CA,C,OTIB:ASP 62:N,CA,C,O,CB,CG,OD1,OD2TIB:VAL 63:N,CA,C,O,CB,CG1,CG2TIB:THR 64:N,CA,C,O,CB,OG1,CG2TIB:GLY 65:N,CA,C,OTIB:PHE 66:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZTIB:ALA 68:N,CA,C,O,CBTIB:ILE 76:N,CA,C,O,CB,CG1,CG2,CD1TIB:VAL 77:N,CA,C,O,CB,CG1,CG2TIB:LEU 78:N,CA,C,O,CB,CG,CD1,CD2TIB:SER 79:N,CA,C,O,CB,OGTIB:ASN 88:N,CA,C,O,CB,CG,OD1,ND2TIB:GLY 91:N,CA,C,OTIB:ASN 92:N,CA,C,O,CB,CG,OD1,ND2TIB:LEU 93:N,CA,C,O,CB,CG,CD1,CD2TIB:ILE 100:N,CA,C,O,CB,CG1,CG2,CD1TIB:ASN 101:N,CA,C,O,CB,CG,OD1,ND2TIB:ASP 102:N,CA,C,O,CB,CG,OD1,OD2TIB:ILE 103:N,CA,C,O,CB,CG1,CG2,CD1TIB:CYS 104:N,CA,C,O,CB,SGTIB:SER 105:N,CA,C,O,CB,OGTIB:GLY 106:N,CA,C,OTIB:CYS 107:N,CA,C,O,CB,SGTIB:SER 116:N,CA,C,O,CB,OGTIB:TRP 117:N,CA,C,O,CB,CG,CD1,CD2,NE1,CE2,CE3,CZ2,CZ3,CH2TIB:SER 119:N,CA,C,O,CB,OGTIB:VAL 120:N,CA,C,O,CB,CG1,CG2TIB:ALA 121:N,CA,C,O,CBTIB:VAL 132:N,CA,C,O,CB,CG1,CG2TIB:ARG 133:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2TIB:GLU 134:N,CA,C,O,CB,CG,CD,OE1,OE2TIB:PRO 136:N,CA,CD,C,O,CB,CGTIB:ARG 139:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2TIB:VAL 140:N,CA,C,O,CB,CG1,CG2TIB:VAL 141:N,CA,C,O,CB,CG1,CG2TIB:PHE 142:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZTIB:VAL 154:N,CA,C,O,CB,CG1,CG2TIB:ALA 155:N,CA,C,O,CBTIB:GLY 156:N,CA,C,OTIB:ALA 157:N,CA,C,O,CBTIB:ASP 158:N,CA,C,O,CB,CG,OD1,OD2TIB:LEU 159:N,CA,C,O,CB,CG,CD1,CD2TIB:ARG 160:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2TIB:GLY 161:N,CA,C,OTIB:ASN 162:N,CA,C,O,CB,CG,OD1,ND2
TIB:GLY 163:N,CA,C,O
TIB:TYR 164:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH
TIB:ASP 165:N,CA,C,O,CB,CG,OD1,OD2
TIB:ILE 166:N,CA,C,O,CB,CG1,CG2,CD1
TIB:ASP 167:N,CA,C,O,CB,CG,OD1,OD2
TIB:VAL 168:N,CA,C,O,CB,CG1,CG2
TIB:PHE 169:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ
TIB:GLY 177:N,CA,C,O
TIB:ASN 178:N,CA,C,O,CB,CG,OD1,ND2
TIB:ARG 179:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2
TIB:ALA 180:N,CA,C,O,CB
TIB:PHE 181:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ
TIB:ALA 182:N,CA,C,O,CB
TIB:GLU 183:N,CA,C,O,CB,CG,CD,OE1,OE2
TIB:PHE 184:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ
TIB:LEU 185:N,CA,C,O,CB,CG,CD1,CD2
TIB:VAL 187:N,CA,C,O,CB,CG1,CG2
TIB:THR 189:N,CA,C,O,CB,OG1,CG2
TIB:GLY 190:N,CA,C,O
TIB:GLY 191:N,CA,C,O
TIB:PRO 207:N,CA,CD,C,O,CB,CG
TIB:PRO 208:N,CA,CD,C,O,CB,CG
TIB:ARG 209:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2
TIB:GLU 210:N,CA,C,O,CB,CG,CD,OE1,OE2
TIB:PHE 211:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ
TIB:GLY 212:N,CA,C,O
TIB:SER 214:N,CA,C,O,CB,OG
TIB:HIS 215:N,CA,C,O,CB,CG,ND1,CD2,CE1,NE2
TIB:SER 216:N,CA,C,O,CB,OG
TIB:GLY 225:N,CA,C,O
TIB:LEU 227:N,CA,C,O,CB,CG,CD1,CD2
TIB:VAL 228:N,CA,C,O,CB,CG1,CG2
TIB:PRO 229:N,CA,CD,C,O,CB,CG
TIB:ILE 241:N,CA,C,O,CB,CG1,CG2,CD1
TIB:ASP 242:N,CA,C,O,CB,CG,OD1,OD2
TIB:ALA 243:N,CA,C,O,CB
TIB:THR 244:N,CA,C,O,CB,OG1,CG2
TIB:PRO 250:N,CA,CD,C,O,CB,CG
TIB:PHE 262:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ
TIB:CYS 268:N,CA,C,O,CB,SGSubset SUB5B:sub5mole.listSubset SUB5B:TIB:3-4,6-7,10-12,15,22-23,25-30,35,37,39,41-42,44-47,51-55,67,69-70,TIB:72,74-75,94-99,108-112,114-115,118,122-126,128-131,135,137-138,TIB:186,188,192-195,213,217-219,223-224,230-231,234-235,238-240,TIB:245,269sub5batom.listSubset SUB5B:
TIB:SER 3:N,CA,C,O,CB,OG
TIB:GLN 4:N,CA,C,O,CB,CG,CD,OE1,NE2
TIB:LEU 6:N,CA,C,O,CB,CG,CD1,CD2
TIB:PHE 7:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ
TIB:PHE 10:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZTIB:ASN 11:N,CA,C,O,CB,CG,OD1,ND2TIB:LEU 12:N,CA,C,O,CB,CG,CD1,CD2TIB:GLN 15:N,CA,C,O,CB,CG,CD,OE1,NE2TIB:CYS 22:N,CA,C,O,CB,SGTIB:GLY 23:N,CA,C,OTIB:ASN 25:N,CA,C,O,CB,CG,OD1,ND2TIB:ASN 26:N,CA,C,O,CB,CG,OD1,ND2TIB:ASP 27:N,CA,C,O,CB,CG,OD1,OD2TIB:ALA 28:N,CA,C,O,CBTIB:PRO 29:N,CA,CD,C,O,CB,CGTIB:ALA 30:N,CA,C,O,CBTIB:THR 35:N,CA,C,O,CB,OG1,CG2TIB:THR 37:N,CA,C,O,CB,OG1,CG2TIB:ASN 39:N,CA,C,O,CB,CG,OD1,ND2TIB:CYS 41:N,CA,C,O,CB,SGTIB:PRO 42:N,CA,CD,C,O,CB,CGTIB:VAL 44:N,CA,C,O,CB,CG1,CG2TIB:GLU 45:N,CA,C,O,CB,CG,CD,OE1,OE2TIB:LYS 46:N,CA,C,O,CB,CG,CD,CE,NZTIB:ALA 47:N,CA,C,O,CBTIB:PHE 51:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZTIB:LEU 52:N,CA,C,O,CB,CG,CD1,CD2TIB:TYR 53:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OHTIB:SER 54:N,CA,C,O,CB,OGTIB:PHE 55:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZTIB:LEU 67:N,CA,C,O,CB,CG,CD1,CD2TIB:LEU 69:N,CA,C,O,CB,CG,CD1,CD2TIB:ASP 70:N,CA,C,O,CB,CG,OD1,OD2TIB:THR 72:N,CA,C,O,CB,OG1,CG2TIB:LYS 74:N,CA,C,O,CB,CG,CD,CE,NZTIB:LEU 75:N,CA,C,O,CB,CG,CD1,CD2TIB:ASN 94:N,CA,C,O,CB,CG,OD1,ND2TIB:PHE 95:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZTIB:ASP 96:N,CA,C,O,CB,CG,OD1,OD2TIB:LEU 97:N,CA,C,O,CB,CG,CD1,CD2TIB:LYS 98:N,CA,C,O,CB,CG,CD,CE,NZTIB:GLU 99:N,CA,C,O,CB,CG,CD,OE1,OE2TIB:ARG 108:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2TIB:GLY 109:N,CA,C,OTIB:HIS 110:N,CA,C,O,CB,CG,ND1,CD2,CE1,NE2TIB:ASP 111:N,CA,C,O,CB,CG,OD1,OD2TIB:GLY 112:N,CA,C,OTIB:THR 114:N,CA,C,O,CB,OG1,CG2TIB:SER 115:N,CA,C,O,CB,OGTIB:ARG 118:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2TIB:ASP 122:N,CA,C,O,CB,CG,OD1,OD2TIB:THR 123:N,CA,C,O,CB,OG1,CG2TIB:LEU 124:N,CA,C,O,CB,CG,CD1,CD2TIB:ARG 125:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2TIB:GLN 126:N,CA,C,O,CB,CG,CD,OE1,NE2TIB:VAL 128:N,CA,C,O,CB,CG1,CG2TIB:GLU 129:N,CA,C,O,CB,CG,CD,OE1,OE2TIB:ASP 130:N,CA,C,O,CB,CG,OD1,OD2TIB:ALA 131:N,CA,C,O,CBTIB:HIS 135:N,CA,C,O,CB,CG,ND1,CD2,CE1,NE2TIB:ASP 137:N,CA,C,O,CB,CG,OD1,OD2TIB:TYR 138:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH
TIB:THR 186:N,CA,C,O,CB,OG1,CG2
TIB:GLN 188:N,CA,C,O,CB,CG,CD,OE1,NE2
TIB:THR 192:N,CA,C,O,CB,OG1,CG2
TIB:LEU 193:N,CA,C,O,CB,CG,CD1,CD2
TIB:TYR 194:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH
TIB:ARG 195:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2
TIB:TYR 213:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH
TIB:SER 217:N,CA,C,O,CB,OG
TIB:PRO 218:N,CA,CD,C,O,CB,CG
TIB:GLU 219:N,CA,C,O,CB,CG,CD,OE1,OE2
TIB:LYS 223:N,CA,C,O,CB,CG,CD,CE,NZ
TIB:SER 224:N,CA,C,O,CB,OG
TIB:VAL 230:N,CA,C,O,CB,CG1,CG2
TIB:THR 231:N,CA,C,O,CB,OG1,CG2
TIB:ASP 234:N,CA,C,O,CB,CG,OD1,OD2
TIB:ILE 235:N,CA,C,O,CB,CG1,CG2,CD1
TIB:ILE 238:N,CA,C,O,CB,CG1,CG2,CD1
TIB:GLU 239:N,CA,C,O,CB,CG,CD,OE1,OE2
TIB:GLY 240:N,CA,C,O
TIB:GLY 245:N,CA,C,O
TIB:LEU 269:N,CA,C,O,CB,OXT,CG,CD1,CD2Subset ACTSITE:actsitemole.listSubset ACTSITE:
TIB:17,21,80-87,89-90,113,143-153,170-176,196-206,221-222,226,246-249,TIB:251-261,263-267actsiteatom.listSubset ACTSITE:
TIB:SER 17:N,CA,C,O,CB,OG
TIB:TYR 21:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH
TIB:PHE 80:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ
TIB:ARG 81:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2
TIB:GLY 82:N,CA,C,O
TIB:SER 83:N,CA,C,O,CB,OG
TIB:ARG 84:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2
TIB:SER 85:N,CA,C,O,CB,OG
TIB:ILE 86:N,CA,C,O,CB,CG1,CG2,CD1
TIB:GLU 87:N,CA,C,O,CB,CG,CD,OE1,OE2
TIB:TRP 89:N,CA,C,O,CB,CG,CD1,CD2,NE1,CE2,CE3,CZ2,CZ3,CH2
TIB:ILE 90:N,CA,C,O,CB,CG1,CG2,CD1
TIB:PHE 113:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ
TIB:THR 143:N,CA,C,O,CB,OG1,CG2
TIB:GLY 144:N,CA,C,O
TIB:HIS 145:N,CA,C,O,CB,CG,ND1,CD2,CE1,NE2
TIB:SER 146:N,CA,C,O,CB,OG
TIB:LEU 147:N,CA,C,O,CB,CG,CD1,CD2
TIB:GLY 148:N,CA,C,O
TIB:GLY 149:N,CA,C,O
TIB:ALA 150:N,CA,C,O,CB
TIB:LEU 151:N,CA,C,O,CB,CG,CD1,CD2
TIB:ALA 152:N,CA,C,O,CB
TIB:THR 153:N,CA,C,O,CB,OG1,CG2
TIB:SER 170:N,CA,C,O,CB,OG
TIB:TYR 171:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH
TIB:GLY 172:N,CA,C,O
TIB:ALA 173:N,CA,C,O,CB
TIB:PRO 174:N,CA,CD,C,O,CB,CG
TIB:ARG 175:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2
TIB:VAL 176:N,CA,C,O,CB,CG1,CG2
TIB:ILE 196:N,CA,C,O,CB,CG1,CG2,CD1
TIB:THR 197:N,CA,C,O,CB,OG1,CG2
TIB:HIS 198:N,CA,C,O,CB,CG,ND1,CD2,CE1,NE2
TIB:THR 199:N,CA,C,O,CB,OG1,CG2
TIB:ASN 200:N,CA,C,O,CB,CG,OD1,ND2
TIB:ASP 201:N,CA,C,O,CB,CG,OD1,OD2
TIB:ILE 202:N,CA,C,O,CB,CG1,CG2,CD1
TIB:VAL 203:N,CA,C,O,CB,CG1,CG2
TIB:PRO 204:N,CA,CD,C,O,CB,CG
TIB:ARG 205:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2
TIB:LEU 206:N,CA,C,O,CB,CG,CD1, CD2
TIB:TRP
221:N,CA,C,O,CB,CG,CD1,CD2,NE1,CE2,CE3,CZ2,CZ3,CH2
TIB:ILE 222:N,CA,C,O,CB,CG1,CG2,CD1
TIB:THR 226:N,CA,C,O,CB,OG1,CG2
TIB:GLY 246:N,CA,C,O
TIB:ASN 247:N,CA,C,O,CB,CG,OD1,ND2
TIB:ASN 248:N,CA,C,O,CB,CG,OD1,ND2
TIB:GLN 249:N,CA,C,O,CB,CG,CD,OE1,NE2
TIB:ASN 251:N,CA,C,O,CB,CG,OD1,ND2
TIB:ILE 252:N,CA,C,O,CB,CG1,CG2,CD1
TIB:PRO 253:N,CA,CD,C,O,CB,CG
TIB:ASP 254:N,CA,C,O,CB,CG,OD1,OD2
TIB:ILE 255:N,CA,C,O,CB,CG1,CG2,CD1
TIB:PRO 256:N,CA,CD,C,O,CB,CG
TIB:ALA 257:N,CA,C,O,CB
TIB:HIS 258:N,CA,C,O,CB,CG,ND1,CD2,CE1,NE2
TIB:LEU 259:N,CA,C,O,CB,CG,CD1,CD2
TIB:TRP
260:N,CA,C,O,CB,CG,CD1,CD2,NE1,CE2,CE3,CZ2,CZ3,CH2
TIB:TYR 261:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH
TIB:GLY 263:N,CA,C,O
TIB:LEU 264:N,CA,C,O,CB,CG,CD1,CD2
TIB:ILE 265:N,CA,C,O,CB,CG1,CG2,CD1
TIB:GLY 266:N,CA,C,O
TIB:THR 267:N,CA,C,O,CB,OG1,CG2Subset RESTX:restxmole.listSubset RESTX:
NEWMODEL:14,16,18-20,31-34,36,38,40,48-50,56-66,68,78-79,88,91-93,NEWMODEL:104-106,120,136,225,227-229,250,262,268restxatom.listSubset RESTX:
NEWMODEL:ALA 14:N,CA,C,O,CB
NEWMODEL:TYR 16:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH
NEWMODEL:ALA 18:N,CA,C,O,CB
NEWMODEL:ALA 19:N,CA,C,O,CB
NEWMODEL:ALA 20:N,CA,C,O,CB
NEWMODEL:GLY 31:N,CA,C,O
NEWMODEL:THR 32:N,CA,C,O,CB,OG1,CG2
NEWMODEL:ASN 33:N,CA,C,O,CB,CG,OD1,ND2
NEWMODEL:ILE 34:N,CA,C,O,CB,CG1,CG2,CD1
NEWMODEL:CYS 36:N,CA,C,O,CB,SGNEWMODEL:GLY 38:N,CA,C,ONEWMODEL:ALA 40:N,CA,C,O,CBNEWMODEL:ASP 48:N,CA,C,O,CB,CG,OD1,OD2NEWMODEL:ALA 49:N,CA,C,O,CBNEWMODEL:THR 50:N,CA,C,O,CB,OG1,CG2NEWMODEL:GLU 56:N,CA,C,O,CB,CG,CD,OE1,OE2NEWMODEL:ASP 57:N,CA,C,O,CB,CG,OD1,OD2NEWMODEL:SER 58:N,CA,C,O,CB,OGNEWMODEL:GLY 59:N,CA,C,ONEWMODEL:VAL 60:N,CA,C,O,CB,CG1,CG2NEWMODEL:GLY 61:N,CA,C,ONEWMODEL:ASP 62:N,CA,C,O,CB,CG,OD1,OD2NEWMODEL:VAL 63:N,CA,C,O,CB,CG1,CG2NEWMODEL:THR 64:N,CA,C,O,CB,OG1,CG2NEWMODEL:GLY 65:N,CA,C,ONEWMODEL:PHE 66:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZNEWMODEL:ALA 68:N,CA,C,O,CBNEWMODEL:LEU 78:N,CA,C,O,CB,CG,CD1,CD2NEWMODEL:SER 79:N,CA,C,O,CB,OGNEWMODEL:ASN 88:N,CA,C,O,CB,CG,OD1,ND2NEWMODEL:GLY 91:N,CA,C,ONEWMODEL:ASN 92:N,CA,C,O,CB,CG,OD1,ND2NEWMODEL:LEU 93:N,CA,C,O,CB,CG,CD1,CD2NEWMODEL:CYS 104:N,CA,C,O,CB,SGNEWMODEL:SER 105:N,CA,C,O,CB,OGNEWMODEL:GLY 106:N,CA,C,ONEWMODEL:VAL 120:N,CA,C,O,CB,CG1,CG2NEWMODEL:PRO 136:N,CA,CD,C,O,CB,CGNEWMODEL:GLY 225:N,CA,C,ONEWMODEL:LEU 227:N,CA,C,O,CB,CG,CD1,CD2NEWMODEL:VAL 228:N,CA,C,O,CB,CG1,CG2NEWMODEL:PRO 229:N,CA,CD,C,O,CB,CGNEWMODEL:PRO 250:N,CA,CD,C,O,CB,CGNEWMODEL:PHE 262:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZNEWMODEL:CYS 268:N,CA,C,O,CB,SG实施例10:提供脂肪酶变体E87K+D254K
按照WO92/05249所述,构建、表达和纯化Humicola lanuginosa脂肪酶变体E87K+D254K。实施例11:脂肪酶-SPEG15000偶联物
按照实施例7所述制备脂肪酶变体E87K+D254K-SPEG偶联物,不同之处在于所用酶为实施例10所述的Humicola lanuginosa脂肪酶变体(E87K+D254K),聚合物是mPEG15000。实施例12:Balb/c小鼠中脂肪酶变体(D87K+D254K)评估为IgG1的免疫原性
通过皮下注射下述物质免疫Balb/c小鼠:i)0.9%(wt/vol)NaCl溶液50μl(对照组,8只小鼠)(对照),ii) 含有25μg Humicola lanuginosa脂肪酶变体(E87K+D254K)蛋白
质的0.9%(wt/vol)NaCl溶液50μl(组1,8只小鼠)(未修饰的脂肪
酶变体),iii)含有在D87K+D254K位置发生了取代、并偶联至经N-琥珀酰亚胺
碳酸酯活化了的mPEG15000上的Humicola lanuginosa脂肪酶变
体的0.9%(wt/vol)NaCl溶液50%(组2,8只小鼠)(脂肪酶-
SPEG15000)。
通过光密度测量,得到每一批号的蛋白质量。免疫接种一周之后且在下次免疫接种之前,从眼部收集血样(200μl)。通过血液凝集和离心得到血清。
采用上述的Balb/c小鼠IgG1 ELISA方法测定IgG1应答。
结果:
需要5次(每周1次)的免疫接种来刺激对未修饰的Humicolalanuginosa变体产生可检测的体液应答。由偶联物(即脂肪酶-SPEG15000)所刺激的抗体滴度为960-1920,与未修饰的HL82-Lipolase所刺激的抗体滴度3840相比,仅低2-4倍(左边的数字)。
结果示于图1中。
对于本领域技术人员来说,显然,根据前述的公开内容,在本发明的实施中,可以有很多改变和修饰,而仍不偏离其精神或范围。因此,本发明的范围应按照下述权利要求的实质内容理解。
序列表(1)一般资料:(i)申请人:
(A)姓名:Novo Nordisk A/S
(B)街道:Novo Alle
(C)城市:Bagsvaerd
(E)国家:丹麦
(F)邮政编码(ZIP):DK-2880
(G)电话:45 4444 8888
(H)传真:45 4449 3256(ii)发明题目:一种被修饰的多肽(iii)序列数:9(iv)计算机可读形式:
(A)介质类型:软盘
(B)计算机:IBM PC可兼容机
(C)操作系统:PC-DOS/MS-DOS
(D)软件:PatentIn Release #1.0,Version #1.30
(EPO)(2)SEQ ID NO:1的资料:(i)序列特征:
(A)长度:840个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(vi)生物来源:
(B)菌株:芽孢杆菌属之种的PD498,NCIMB No.40484(ix)特征:
(A)名称/关键词:CDS
(B)位置:1..840(xi)序列描述:SEQ ID NO:1:TGG TCA CCG AAT GAC CCT TAC TAT TCT GCT TAC CAG TAT GGA CCA CAA 48Trp Ser Pro Asn Asp Pro Tyr Tyr Ser Ala Tyr Gln Tyr Gly Pro Gln1 5 10 15AAC ACC TCA ACC CCT GCT GCC TGG GAT GTA ACC CGT GGA AGC AGC ACT 96Asn Thr Ser Thr Pro Ala Ala Trp Asp Val Thr Arg Gly Ser Ser Thr
20 25 30CAA ACG GTG GCG GTC CTT GAT TCC GGA GTG GAT TAT AAC CAC CCT GAT 144Gln Thr Val Ala Val Leu Asp Ser Gly Val Asp Tyr Asn His Pro Asp
35 40 45CTT GCA AGA AAA GTA ATA AAA GGG TAC GAC TTT ATC GAC AGG GAC AAT 192Leu Ala Arg Lys Val Ile Lys Gly Tyr Asp Phe Ile Asp Arg Asp Asn
50 55 60AAC CCA ATG GAT CTT AAC GGA CAT GGT ACC CAT GTT GCC GGT ACT GTT 240Asn Pro Met Asp Leu Asn Gly His Gly Thr His Val Ala Gly Thr Val65 70 75 80GCT GCT GAT ACG AAC AAT GGA ATT GGC GTA GCC GGT ATG GCA CCA GAT 288Ala Ala Asp Thr Asn Asn Gly Ile Gly Val Ala Gly Met Ala Pro Asp
85 90 95ACG AAG ATC CTT GCC GTA CGG GTC CTT GAT GCC AAT GGA AGT GGC TCA 336Thr Lys Ile Leu Ala Val Arg Val Leu Asp Ala Asn Gly Ser Gly Ser
100 105 110CTT GAC AGC ATT GCC TCA GGT ATC CGC TAT GCT GCT GAT CAA GGG GCA 384Leu Asp Ser Ile Ala Ser Gly Ile Arg Tyr Ala Ala Asp Gln Gly Ala
115 120 125AAG GTA CTC AAC CTC TCC CTT GGT TGC GAA TGC AAC TCC ACA ACT CTT 432Lys Val Leu Asn Leu Ser Leu Gly Cys Glu Cys Asn Ser Thr Thr Leu
130 135 140AAG AGT GCC GTC GAC TAT GCA TGG AAC AAA GGA GCT GTA GTC GTT GCT 480Lys Ser Ala Val Asp Tyr Ala Trp Asn Lys Gly Ala Val Val Val Ala145 150 155 160GCT GCA GGG AAT GAC AAT GTA TCC CGT ACA TTC CAA CCA GCT TCT TAC 528Ala Ala Gly Asn Asp Asn Val Ser Arg Thr Phe Gln Pro Ala Ser Tyr
165 170 175CCT AAT GCC ATT GCA GTA GGT GCC ATT GAC TCC AAT GAT CGA AAA GCA 576Pro Asn Ala Ile Ala Val Gly Ala Ile Asp Ser Asn Asp Arg Lys Ala
180 185 190TCA TTC TCC AAT TAC GGA ACG TGG GTG GAT GTC ACT GCT CCA GGT GTG 624Ser Phe Ser Asn Tyr Gly Thr Trp Val Asp Val Thr Ala Pro Gly Val
195 200 205AAC ATA GCA TCA ACC GTT CCG AAT AAT GGC TAC TCC TAC ATG TCT GGT 672Asn Ile Ala Ser Thr Val Pro Asn Asn Gly Tyr Ser Tyr Met Ser Gly
210 215 220ACG TCC ATG GCA TCC CCT CAC GTG GCC GGT TTG GCT GCT TTG TTG GCA 720Thr Ser Met Ala Ser Pro His Val Ala Gly Leu Ala Ala Leu Leu Ala225 230 235 240AGT CAA GGT AAG AAT AAC GTA CAA ATC CGC CAG GCC ATT GAG CAA ACC 768Ser Gln Gly Lys Asn Asn Val Gln Ile Arg Gln Ala Ile Glu Gln Thr
245 250 255GCC GAT AAG ATC TCT GGC ACT GGA ACA AAC TTC AAG TAT GGT AAA ATC 816Ala Asp Lys Ile Ser Gly Thr Gly Thr Asn Phe Lys Tyr Gly Lys Ile
260 265 270AAC TCA AAC AAA GCT GTA AGA TAC 840Asn Ser Asn Lys Ala Val Arg Tyr
275 280(2)SEQ ID NO:2的资料:(i)序列特征:
(A)长度:280个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性(ii)分子类型:蛋白质(xi)序列描述:SEQ ID NO:2:Trp Ser Pro Asn Asp Pro Tyr Tyr Ser Ala Tyr Gln Tyr Gly Pro Gln1 5 10 15Asn Thr Ser Thr Pro Ala Ala Trp Asp Val Thr Arg Gly Ser Ser Thr
20 25 30Gln Thr Val Ala Val Leu Asp Ser Gly Val Asp Tyr Asn His Pro Asp
35 40 45Leu Ala Arg Lys Val Ile Lys Gly Tyr Asp Phe Ile Asp Arg Asp Asn
50 55 60Asn Pro Met Asp Leu Asn Gly His Gly Thr His Val Ala Gly Thr Val65 70 75 80Ala Ala Asp Thr Asn Asn Gly Ile Gly Val Ala Gly Met Ala Pro Asp
85 90 95Thr Lys Ile Leu Ala Val Arg Val Leu Asp Ala Asn Gly Ser Gly Ser
100 105 110Leu Asp Ser Ile Ala Ser Gly Ile Arg Tyr Ala Ala Asp Gln Gly Ala
115 120 125Lys Val Leu Asn Leu Ser Leu Gly Cys Glu Cys Asn Ser Thr Thr Leu
130 135 140Lys Ser Ala Val Asp Tyr Ala Trp Asn Lys Gly Ala Val Val Val Ala145 150 155 160Ala Ala Gly Asn Asp Asn Val Ser Arg Thr Phe Gln Pro Ala Ser Tyr
165 170 175Pro Asn Ala Ile Ala Val Gly Ala Ile Asp Ser Asn Asp Arg Lys Ala
180 185 190Ser Phe Ser Asn Tyr Gly Thr Trp Val Asp Val Thr Ala Pro Gly Val
195 200 205Asn Ile Ala Ser Thr Val Pro Asn Asn Gly Tyr Ser Tyr Met Ser Gly
210 215 220Thr Ser Met Ala Ser Pro His Val Ala Gly Leu Ala Ala Leu Leu Ala225 230 235 240Ser Gln Gly Lys Asn Asn Val Gln Ile Arg Gln Ala Ile Glu Gln Thr
245 250 255Ala Asp Lys Ile Ser Gly Thr Gly Thr Asn Phe Lys Tyr Gly Lys Ile
260 265 270Asn Ser Asn Lys Ala Val Arg Tyr
275 280(2)SEQ ID NO:3的资料:(i)序列特征:
(A)长度:269个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:蛋白质(vi)生物来源:
(B)菌株:迟缓芽孢杆菌(xi)序列描述:SEQ ID NO:1:Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala1 5 10 15His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp
20 25 30Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser
35 40 45Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr
50 55 60His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu65 70 75 80Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala
85 90 95Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala
100 105 110Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser
115 120 125Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly
130 135 140Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser145 150 155 160Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln
165 170 175Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile
180 185 190Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr Tyr
195 200 205Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala
210 215 220Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile225 230 235 240Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu
245 250 255Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg
260 265(2)SEQ ID NO:4的资料:(i)序列特征:
(A)长度:344个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:蛋白质(vi)生物来源:
(B)菌株:Arthromyces ramosus(xi)序列描述:SEQ ID NO:1:Gln Gly Pro Gly Gly Gly Gly Gly Ser Val Thr Cys Pro Gly Gly Gln1 5 10 15Ser Thr Ser Asn Ser Gln Cys Cys Val Trp Phe Asp Val Leu Asp Asp
20 25 30Leu Gln Thr Asn Phe Tyr Gln Gly Ser Lys Cys Glu Ser Pro Val Arg
35 40 45Lys Ile Leu Arg Ile Val Phe His Asp Ala Ile Gly Phe Ser Pro Ala
50 55 60Leu Thr Ala Ala Gly Gln Phe Gly Gly Gly Gly Ala Asp Gly Ser Ile65 70 75 80Ile Ala His Ser Asn Ile Glu Leu Ala Phe Pro Ala Asn Gly Gly Leu
85 90 95Thr Asp Thr Ile Glu Ala Leu Arg Ala Val Gly Ile Asn His Gly Val
100 105 110Ser Phe Gly Asp Leu Ile Gln Phe Ala Thr Ala Val Gly Met Ser Asn
115 120 125Cys Pro Gly Ser Pro Arg Leu Glu Phe Leu Thr Gly Arg Ser Asn Ser
130 135 140Ser Gln Pro Ser Pro Pro Ser Leu Ile Pro Gly Pro Gly Asn Thr Val145 150 155 160Thr Ala Ile Leu Asp Arg Met Gly Asp Ala Gly Phe Ser Pro Asp Glu
165 170 175Val Val Asp Leu Leu Ala Ala His Ser Leu Ala Ser Gln Glu Gly Leu
180 185 190Asn Ser Ala Ile Phe Arg Ser Pro Leu Asp Ser Thr Pro Gln Val Phe
195 200 205Asp Thr Gln Phe Tyr Ile Glu Thr Leu Leu Lys Gly Thr Thr Gln Pro
210 215 220Gly Pro Ser Leu Gly Phe Ala Glu Glu Leu Ser Pro Phe Pro Gly Glu225 230 235 240Phe Arg Met Arg Ser Asp Ala Leu Leu Ala Arg Asp Ser Arg Thr Ala
245 250 255Cys Arg Trp Gln Ser Met Thr Ser Ser Asn Glu Val Met Gly Gln Arg
260 265 270Tyr Arg Ala Ala Met Ala Lys Met Ser Val Leu Gly Phe Asp Arg Asn
275 280 285Ala Leu Thr Asp Cys Ser Asp Val Ile Pro Ser Ala Val Ser Asn Asn
290 295 300Ala Ala Pro Val Ile Pro Gly Gly Leu Thr Val Asp Asp Ile Glu Val305 310 315 320Ser Cys Pro Ser Glu Pro Phe Pro Glu Ile Ala Thr Ala Ser Gly Pro
325 330 335Leu Pro Ser Leu Ala Pro Ala Pro
340(2)SEQ ID NO:5的资料:(i)序列特征:
(A)长度:876个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(vi)生物来源:
(B)菌株:Humicola lanuginosa DSM4109(ix)特征:
(A)名称/关键词:信号肽
(B)位置:1..66 (ix)特征:
(A)名称/关键词:成熟肽
(B)位置:67..876(ix)特征:
(D)名称/关键词:CDS
(E)位置:1..876(xi)序列描述:SEQ ID NO:5:ATG AGG AGC TCC CTT GTG CTG TTC TTT GTC TCT GCG TGG ACG GCC TTG 48Met Arg Ser Ser Leu Val Leu Phe Phe Val Ser Ala Trp Thr Ala Leu-22 -20 -15 -10GCC AGT CCT ATT CGT CGA GAG GTC TCG CAG GAT CTG TTT AAC CAG TTC 96Ala Ser Pro Ile Arg Arg Glu Val Ser Gln Asp Leu Phe Asn Gln Phe
-5 1 5 10AAT CTC TTT GCA CAG TAT TCT GCA GCC GCA TAC TGC GGA AAA AAC AAT 144Asn Leu Phe Ala Gln Tyr Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn
15 20 25GAT GCC CCA GCT GGT ACA AAC ATT ACG TGC ACG GGA AAT GCC TGC CCC 192Asp Ala Pro Ala Gly Thr Asn Ile Thr Cys Thr Gly Asn Ala Cys Pro
30 35 40GAG GTA GAG AAG GCG GAT GCA ACG TTT CTC TAC TCG TTT GAA GAC TCT 240Glu Val Glu Lys Ala Asp Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser
45 50 55GGA GTG GGC GAT GTC ACC GGC TTC CTT GCT CTC GAC AAC ACG AAC AAA 288Gly Val Gly Asp Val Thr Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys
60 65 70TTG ATC GTC CTC TCT TTC CGT GGC TCT CGT TCC ATA GAG AAC TGG ATC 336Leu Ile Val Leu Ser Phe Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile75 80 85 90GGG AAT CTT AAC TTC GAC TTG AAA GAA ATA AAT GAC ATT TGC TCC GGC 384Gly Asn Leu Asn Phe Asp Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly
95 100 105TGC AGG GGA CAT GAC GGC TTC ACT TCG TCC TGG AGG TCT GTA GCC GAT 432Cys Arg Gly His Asp Gly Phe Thr Ser Ser Trp Arg Ser Val Ala Asp
110 115 120ACG TTA AGG CAG AAG GTG GAG GAT GCT GTG AGG GAG CAT CCC GAC TAT 480Thr Leu Arg Gln Lys Val Glu Asp Ala Val Arg Glu His Pro Asp Tyr
125 130 135CGC GTG GTG TTT ACC GGA CAT AGC TTG GGT GGT GCA TTG GCA ACT GTT 528Arg Val Val Phe Thr Gly His Ser Leu Gly GLy Ala Leu Ala Thr Val
140 145 150GCC GGA GCA GAC CTG CGT GGA AAT GGG TAT GAT ATC GAC GTG TTT TCA 576Ala Gly Ala Asp Leu Arg Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser155 160 165 170TAT GGC GCC CCC CGA GTC GGA AAC AGG GCT TTT GCA GAA TTC CTG ACC 624Tyr Gly Ala Pro Arg Val Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr
175 180 185GTA CAG ACC GGC GGA ACA CTC TAC CGC ATT ACC CAC ACC AAT GAT ATT 672Val Gln Thr Gly Gly Thr Leu Tyr Arg Ile Thr His Thr Asn Asp Ile
190 195 200GTC CCT AGA CTC CCG CCG CGC GAA TTC GGT TAC AGC CAT TCT AGC CCA 720Val Pro Arg Leu Pro Pro Arg Glu Phe Gly Tyr Ser His Ser Ser Pro
205 210 215GAG TAC TGG ATC AAA TCT GGA ACC CTT GTC CCC GTC ACC CGA AAC GAT 768Glu Tyr Trp Ile Lys Ser Gly Thr Leu Val Pro Val Thr Arg Asn Asp
220 225 230ATC GTG AAG ATA GAA GGC ATC GAT GCC ACC GGC GGC AAT AAC CAG CCT 816Ile Val Lys Ile Glu Gly Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro235 240 245 250AAC ATT CCG GAT ATC CCT GCG CAC CTA TGG TAC TTC GGG TTA ATT GGG 864Asn Ile Pro Asp Ile Pro Ala His Leu Trp Tyr Phe Gly Leu Ile Gly
255 260 265ACA TGT CTT TAG 876Thr Cys Leu *
270(2)SEQ ID NO:6的资料:(i)序列特征:
(A)长度:292个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性(ii)分子类型:蛋白质(xi)序列描述:SEQ ID NO:2:Met Arg Ser Ser Leu Val Leu Phe Phe Val Ser Ala Trp Thr Ala Leu-22 -20 -15 -10Ala Ser Pro Ile Arg Arg Glu Val Ser Gln Asp Leu Phe Asn Gln Phe
-5 1 5 10Asn Leu Phe Ala Gln Tyr Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn
15 20 25Asp Ala Pro Ala Gly Thr Asn Ile Thr Cys Thr Gly Asn Ala Cys Pro
30 35 40Glu Val Glu Lys Ala Asp Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser
45 50 55Gly Val Gly Asp Val Thr Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys
60 65 70Leu Ile Val Leu Ser Phe Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile75 80 85 90Gly Asn Leu Asn Phe Asp Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly
95 100 105Cys Arg Gly His Asp Gly Phe Thr Ser Ser Trp Arg Ser Val Ala Asp
110 115 120Thr Leu Arg Gln Lys Val Glu Asp Ala Val Arg Glu His Pro Asp Tyr
125 130 135Arg Val Val Phe Thr Gly His Ser Leu Gly Gly Ala Leu Ala Thr Val
140 145 150Ala Gly Ala Asp Leu Arg Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser155 160 165 170Tyr Gly Ala Pro Arg Val Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr
175 180 185Val Gln Thr Gly Gly Thr Leu Tyr Arg Ile Thr His Thr Asn Asp Ile
190 195 200Val Pro Arg Leu Pro Pro Arg Glu Phe Gly Tyr Ser His Ser Ser Pro
205 210 215Glu Tyr Trp Ile Lys Ser Gly Thr Leu Val Pro Val Thr Arg Asn Asp
220 225 230Ile Val Lys Ile Glu Gly Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro235 240 245 250Asn Ile Pro Asp Ile Pro Ala His Leu Trp Tyr Phe Gly Leu Ile Gly
255 260 265Thr Cys Leu *
270(2)SEQ ID NO:7的资料:(i)序列特征:
(A)长度:32个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:其它核酸
(A)描述:/desc=“R28K oligo”(xi)序列描述:SEQ ID NO:7:gggatgtaac caagggaagc agcactcaaa cg 32(2)SEQ ID NO:8的资料:(i)序列特征:
(A)长度:27个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:其它核酸
(A)描述:/desc=“R62K oligo” (xi)序列描述:SEQ ID NO:8:cgactttatc gataaggaca ataaccc 27(2)SEQ ID NO:9的资料:(i)序列特征:
(A)长度:27个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:其它核酸
(A)描述:/desc=“R169K oligo”(xi)序列描述:SEQ ID NO:9:caatgtatcc aaaacgttcc aaccagc 27
Claims (35)
1.一种多肽-聚合物偶联物,其具有:
a)一个或多个额外的聚合分子偶联至多肽上,所述多肽已被修饰,使得与相应亲本多肽上的可用连接基团数相比,该多肽表面上的连接基团数增加了,和/或
b)少一个或多个聚合分子偶联至多肽上,所述多肽已被修饰,使得与相应亲本多肽上的可用连接基团数相比,该多肽的功能性位点处或附近的连接基团数减少了。
2.根据权利要求1的偶联物,与由相应亲本酶制得的偶联物上的聚合分子数相比,其有1-25个、优选1-10个额外的聚合分子偶联到该多肽的表面上。
3.根据权利要求1和2的偶联物,其中所述额外连接基团是赖氨酸残基形式的氨基,或者是天冬氨酸或谷氨酸残基形式的羧基。
4.根据权利要求1-3中任何一项的偶联物,其中所述的额外连接基团是通过氨基酸残基的保守性取代,如精氨酸至赖氨酸的取代而制得的。
5.根据权利要求1-3中任何一项的偶联物,其中所述的额外连接基团是通过氨基酸的保守性取代、如天冬酰胺至天冬氨酸/谷氨酸的取代或者谷氨酰胺至天冬氨酸/谷氨酸的取代而制得的。
6.根据权利要求1-5中任何一项的偶联物,其中添加的连接基团所处的位置距离功能性位点5以上,优选8以上,更优选10以上。
7.根据权利要求1的偶联物,其特征在于,与在相应亲本多肽基础上制得的偶联物上的聚合分子数相比,所述偶联物上偶联到多肽的功能性位点处或者附近的聚合分子要少1-25个、优选1-10个。
8.根据权利要求7的偶联物,其中被除去的连接基团是赖氨酸残基形式的氨基,或者是天冬氨酸或谷氨酸残基形式的羧基。
9.根据权利要求7或8的偶联物,其中连接基团的除去是通过氨基的保守性取代,如赖氨酸至精氨酸的取代而达到的。
10.根据权利要求7或8的偶联物,其中连接基团的除去是通过羧基的保守性取代,如天冬氨酸/谷氨酸至天冬酰胺的取代或者天冬氨酸/谷氨酸至谷氨酰胺的取代而达到的。
11.根据权利要求1-10中任何一项的偶联物,其中被除去的连接基团所处的位置距离功能性位点5以内,优选8以内,更优选10以内。
12.根据权利要求1-11中任何一项的偶联物,其中所述连接基团广泛分布。
13.根据权利要求1-12的偶联物,其中所述偶联物的亲本多肽部分的分子量为1-100kDa,优选15-100kDa。
14.根据权利要求13的偶联物,其中所述偶联物的亲本多肽部分的分子量为1-35kDa。
15.根据权利要求14的偶联物,其中所述亲本多肽是选自下述的一种酶:氧化还原酶,包括漆酶和超氧化物歧化酶(SOD);水解酶,包括蛋白酶,特别是枯草杆菌蛋白酶,和脂解酶;转移酶,包括转谷氨酰胺酶(TG酶);异构酶,包括蛋白质二硫键异构酶(PDI)。
16.根据权利要求15的偶联物,其中所述亲本酶是PD498,Savinase,BPN’,蛋白酶K,蛋白酶R,枯草杆菌蛋白酶DY,Lion Y,Rennilase,JA16,Alcalase或者Humicola lanuginosa脂肪酶,如Lipolase。
17.根据权利要求16的偶联物,其中所述偶联物的酶部分是具有下述一或多个取代的PD498变体:R51K,R62K,R121K,R169K,R250K,R28K,R190K,P6K,Y7K,S9K,A10K,Y11K,Q12K,D43K,Y44K,N45K,N65K,G87K,I88K,N209K,A211K,N216K,N217K,G218K,Y219K,S220K,Y221K,G262K。
18.根据权利要求17的偶联物,其具有下述突变之一:R28K+R62K,R28K+R169K,R62K+R169K,R28K+R69K+R169K。
19.根据权利要求16的偶联物,其中所述偶联物的酶部分是具有下述一或多个取代的Savinase变体:R10K,R19K,R45K,R145K,R170K,R186K,R247K,K94R,P5K,P14K,T22K,T38K,H39K,P40K,L42K,L75K,N76K,L82K,P86K,S103K,V104K,S105K,A108K,A133K,T134K,L135K,Q137K,N140K,N173K,N204K,Q206K,G211K,S212K,T213K,A215K,S216K,N269K。
20.根据权利要求16的偶联物,其中所述偶联物的酶部分是具有下述一或多个取代的Humicola lanuginosa脂肪酶变体:R133K,R139K,R160K,R179K,R209K,R118K,R125K,A18K,G31K,T32K,N33K,G38K,A40K,D48K,T50K,E56K,D57K,S58K,G59K,V60K,G61K,D62K,T64K,L78K,E87K,N88K,G91K,N92K,L93K,S105K,G106K,V120K,P136K,G225K,L227K,V228K,P229K,P250K,D254K,F262K。
21.根据权利要求20的偶联物,其具有下述突变:E87K+D254K。
22.根据权利要求1-21中任何一项的偶联物,其中偶联至多肤上的聚合分子的分子量为1-60kDa,特别是1-35kDa,尤其是3-25kDa。
23.根据权利要求22的偶联物,其中所述聚合分子选自包含天然或合成同聚物和异聚物的组中,该组包括含下述的合成聚合分子:分支PEG、聚乙烯醇(PVA)、聚羧酸、聚(乙烯吡咯烷酮)和聚D,L-氨基酸,或者含下述的天然存在的聚合分子;葡聚糖,包括羧甲基葡聚糖,和纤维素,如甲基纤维素、羧甲基纤维素、乙基纤维素、羟乙基纤维素、羟丙基纤维素,以及聚氨基葡糖的水解产物,淀粉,如羟乙基淀粉、羟丙基淀粉,糖原,琼脂糖,瓜尔胶,菊粉,支链淀粉,合成生物聚合胶,角叉菜胶,果胶和藻酸。
24.制备改良的多肽-聚合物偶联物的方法,包括以下步骤:a)鉴定出位于所述亲本多肽的3D结构表面上的氨基酸残基,b)选择出所述待突变的亲本多肽的所述3D结构表面上的目标氨基酸残基,c)i)用具有合适连接基团的氨基酸残基取代步骤b)中选定的一个或多个氨基酸残基或者在所选位点插入所述氨基酸残基,和/或
ii)取代或者删除步骤b)中选定的、功能性位点处或者附近的一个或多个氨基酸残基,d)将聚合分子偶联到已突变的多肽上。
25.根据权利要求24的方法,其中步骤a)中所指的位于多肽表面上的氨基酸残基的鉴定是利用计算机程序分析所述亲本多肽3D结构进行的。
26.根据权利要求24的方法,其中步骤b)包括选择出亲本多肽表面上的精氨酸或赖氨酸残基。
27.根据权利要求24的方法,其中,在步骤c)中,用赖氨酸残基取代步骤b)中鉴定出的一个或多个精氨酸残基。
28.根据权利要求27的方法,其中被取代的精氨酸残基所处的位置距离功能性位点5以上,优选8以上,特别是10以上。
29.根据权利要求24-28中任何一项的方法,其中将步骤c)中制得的多肽与聚合分子偶联。
30.权利要求1-23中的偶联物在降低工业产品变应原性中的用途。
31.权利要求1-23中的偶联物在降低药物免疫原性中的用途。
32.含有权利要求1-23中任何一项的偶联物以及工业产品中所用成分的一种组合物。
33.根据权利要求32的组合物,其中所述工业产品是洗涤剂,如洗衣、洗盘子或硬表面清洁产品,或者是食品或饲料产品。
34.根据权利要求32的组合物,其中含有权利要求1-22中任何一项的偶联物以及皮肤护理产品中使用的成分。
35.含有权利要求1-23中任何一项的偶联物以及药物中所用成分的组合物。
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- 1998-02-06 CA CA002279986A patent/CA2279986A1/en not_active Abandoned
- 1998-02-06 WO PCT/DK1998/000046 patent/WO1998035026A1/en not_active Application Discontinuation
- 1998-02-06 CN CN98802364A patent/CN1246891A/zh active Pending
- 1998-02-06 JP JP53358498A patent/JP2001511162A/ja active Pending
- 1998-02-06 EP EP98901327A patent/EP1017794A1/en not_active Ceased
- 1998-02-06 AU AU57495/98A patent/AU740207B2/en not_active Ceased
- 1998-02-17 US US09/024,532 patent/US6245901B1/en not_active Expired - Fee Related
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2000
- 2000-11-02 US US09/705,185 patent/US6623950B1/en not_active Expired - Fee Related
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2003
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Cited By (2)
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CN108135966A (zh) * | 2015-08-31 | 2018-06-08 | 汉阳大学校产学协力团 | 包含腺病毒蛋白vi衍生肽的用于细胞内递送的组合物和包含它的抗癌药物组合物 |
CN108135966B (zh) * | 2015-08-31 | 2021-12-28 | 基因药物株式会社 | 包含腺病毒蛋白vi衍生肽的用于细胞内递送的组合物和包含它的抗癌药物组合物 |
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WO1998035026A1 (en) | 1998-08-13 |
CA2279986A1 (en) | 1998-08-13 |
AU740207B2 (en) | 2001-11-01 |
US6623950B1 (en) | 2003-09-23 |
US20050079593A1 (en) | 2005-04-14 |
EP1017794A1 (en) | 2000-07-12 |
AU5749598A (en) | 1998-08-26 |
JP2001511162A (ja) | 2001-08-07 |
US6245901B1 (en) | 2001-06-12 |
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