CN1663960A - 高效表达重组人胰岛素原及其类似物的新型c肽 - Google Patents
高效表达重组人胰岛素原及其类似物的新型c肽 Download PDFInfo
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- CN1663960A CN1663960A CN 200410007170 CN200410007170A CN1663960A CN 1663960 A CN1663960 A CN 1663960A CN 200410007170 CN200410007170 CN 200410007170 CN 200410007170 A CN200410007170 A CN 200410007170A CN 1663960 A CN1663960 A CN 1663960A
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Abstract
本发明涉及一种用于重组制备胰岛素原及其类似物的新型C肽。应用本发明提供的新型C肽构建重组表达胰岛素原,具有高表达量、易纯化和高回收率等优点,适用于重组胰岛素及其类似物的制备。
Description
本发明提供了一种用重组技术制备胰岛素原及其类似物的新型C肽。
人体内成熟的活性胰岛素分子量是5808道尔顿,由A链和B链两条氨基酸肽链组成。A链有21个氨基酸,B链有30个氨基酸。A-B链之间有两个二硫键相连。胰岛素最初是以一条单链分子的前体形式合成的,即胰岛素原(Proinsulin)。胰岛素原是由B-C-A三条肽链连接而成,C肽的C末端通过两个碱性氨基酸残基(Lys Arg)与胰岛素A链的N末端相连,N末端通过另外两个碱性氨基酸残基(Arg Arg)与胰岛素B链的C末端相连。在细胞高尔基体中,胰岛素原被贮存在储存颗粒内,并在肽酶的催化下,切去C肽而形成活性胰岛素。活性胰岛素与C肽以相等分子分泌进入血液。由此可见,C肽的存在是胰岛素实现其正确空间结构并保证生物活性的必然条件。
以往认为,C肽含有使胰岛素二硫键正确配对的足够结构信息,没有C肽将A链和B链连接在一起,活性胰岛素就无法使其两条链弯曲成适当的角度以形成二硫键的连接。近期在研究胰岛素的结构与生物功能的关系中发现,C肽在胰岛素A、B链正确配对时,可能只起柔性连结肽作用,使得双分子反应变为分子内反应。单独的A或B链本身就具有一定的二级结构,在溶液中二者按一定的方式相互作用正确配对,从而导致重组胰岛素的产率较高。用蛋白二硫键异构酶处理二硫键错接的胰岛素衍生物,可以重新生成胰岛素,C肽对这一反应无影响。与胰岛素原相似,用简单的交联剂连接A、B链的交联胰岛素,也可以把二硫键错接的产物以很高的产率生成二硫键正确连接的交联胰岛素。这些化学交联剂与胰岛素原中的C肽不同,它们的结构简单而种类繁多,不同的化学交联剂中是不可能含有胰岛素结构信息的。因此,形成正确连接二硫键的结构信息只能存在于胰岛素的A及B链中。既然A、B链包含了足够的使二硫键正确配对的信息,然而基因工程重组胰岛素原和天然人胰岛素原的A及B链是经细胞一次性表达并连接在一起的肽链,A、B链之间必须串联一个一定长度的肽段把两者的空间位置撑开到适当程度,才能形成由二硫键正确连接的胰岛素。由此可见,C肽的存在、长短将对A、B链的重组产生影响。以往文献和相关专利提供了胰岛素A、B、C链的串联表达方式,即B-C-A,在每个肽链的连接区设计含胰蛋白酶敏感酶切位点,如Arg-Glu、Arg-Gly。
本发明还涉及下面一个问题,即C肽的氨基酸序列对于A链和B链的正确折叠具有怎样的影响?天然人胰岛素原的C肽由35个氨基酸组成,基本序列为:NH-Arg-Arg-Glu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-Lys-Arg-COO。本专利将N端第4-12位氨基酸(斜体部分)用组氨酸替换后,对A链和B链的正确折叠无明显影响。新型C肽N端第1位氨基酸用于与B链的B(30)Thr和相连形成Arg-Arg-Glu的胰蛋白酶切位点。发现第12-35位氨基酸被替换或缺失缩短后,A链和B链无法则形成正确的折叠或折叠效率显著降低。本发明利用这些特点,对天然人C肽的N端第4-12位氨基酸(Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val)进行了氨基酸的替换改造。改造的目的在于提高A、B链间二硫键正确折叠和胰岛素原的重组表达及纯化效率。
本专利所发明的新型利于更便捷高效地获得基因工程重组胰岛素,涉及到不同给药间隔的快速、中效和长效胰岛素制品的重组制备。其中包括了各种不同的人胰岛素类似物,如甘精胰岛素、谷甘胰岛素、赖脯胰岛素、三精胰岛素等。
选择对天然人C肽的N末端4-12位氨基酸进行4-8个组氨酸替换形成本发明的新型C肽,序列如下:NH2-Glu-(0-4)X-(4-8)His-(0-4)Y-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-Lys-Arg-CooH。序列中X、Y为任意氨基酸,(4-8)His定义为4到8个连续的组氨酸序列,(0-4)X定义为0到4个连续的任意氨基酸序列,(0-4)Y定义为0到4个连续的任意氨基酸序列。替换组氨酸后的新型C肽由33个氨基酸组成。该新型C肽具有二个创新特点:其一是不影响人胰岛素原及其类似物的空间结构,与天然C肽具有相同的作用,保证了A链和B链的正确折叠;其二是利用4到8个连续的组氨酸序列,对原核细胞及真核细胞重组表达的人胰岛素原及其类似物等蛋白质可用金属离子镍鏊和层析纯化,在经胰蛋白酶或/和羧肽酶B酶切后的新型C肽、未酶切及酶切不彻底带C肽的片段,可利用金属离子镍鏊和层析去除,从而显著提高相应活性胰岛素的纯度和回收率。
对本专利上述方案都可以进行多种多样的改动,最显著的改动是对天然人C肽的N末端4-12位氨基酸组氨酸以外的替换,以利用其它亲和层析或非亲和层析方法制备人胰岛素及其类似物。
以下的实例用于理解及如何实施本发明,这些实例仅仅在一个侧面说明应用本发明的优点,决不对本发明所涉及的范围进行限制。
实例1.用于原核细胞表达的含新型C肽重组甘精胰岛素原
基因序列设计
甘精胰岛素结构为20A-Gly21和30B-Arg31-Arg32,即将人胰岛素A链的第21位Asn替换成Gly,B链第30位氨基酸后加入2个Arg而成。重组甘精胰岛素原由A(21)链、B(32)链和新型C(33)肽共96个氨基酸组成。在B链和A链之间含5个组氨酸(Glu1-Val2-Pro3-His(4-8)-C肽)的新型C肽(由33个氨基酸组成),并在B链前加入由10个氨基酸组成的疏水性引导肽。最终的表达产物为96个氨基酸组成的融合重组甘精胰岛素原,线性序列为Fus(10)-B(32)-C(33)-A(21)。
重组甘精胰岛素原各组成部分的氨基酸序列如下:
Seq1:Met Ala Thr Thr Ser Thr Ala Thr Thr Arg
Seq2:Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys
Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg
Seq3:Glu Ala Glu His His His His His Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly
Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg
Seq4:Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys
Gly
其中,Seq1为引导肽的10个氨基酸序列。Seq2为B链的32个氨基酸序列。Seq3为新型C肽的33个氨基酸序列,是将人胰岛素C肽的6-10位氨基酸Asp-Leu-Gln-Val-Gly由串联的5个His替换而成。Seq4为A链的21个的氨基酸序列。
根据上述重组甘精胰岛素原的Fus(10)-B(32)-C(33)-A(21)氨基酸序列,按大肠杆菌偏爱密码子设计出对应的核苷酸序列,分别在其5’端引入限制性核酸内切酶Nde I酶切识别位点“CTAATG”,3’端引入终止密码子TAA和限制性核酸内切酶BamH I酶切识别位点“GGATCC”。甘精胰岛素原的cDNA序列如下:
Seq5:5’-CTA ATG GCT ACA ACC TCT ACA GCT ACC ACA CGT TTC GTT AAC
CAG CAC CTG TGC GGT TCT CAC CTG GTT GAA GCT CTG TAC CTG
GTT TGC GGT GAA CGT GGT TTC TTC TAC ACC CCG AAA ACC CGT
CGT GAA GTT CCG CAC CAT CAC CAT CAC CAG GTT GAA CTG GGT
GGT GGT CCG GGT GCT GGT TCT CTG CAG CCG CTG GCT CTG GAA
GGT TCT CTG CAG AAA CGT GGT ATC GTT GAA CAG TGC TGC ACC
TCT ATC TGC TCT CTG TAC CAG CTG GAA AAC TAC TGC GGT TAA
GGA TCC-3’
对上述设计的核苷酸序列进行全基因人工合成并测序正确后用于进行原核表达载体构建。
实例2.含新型C肽重组甘精胰岛素原大肠杆菌
表达工程菌的获得
一、重组甘精胰岛素原表达质粒的构建和鉴定
将全基因人工合成的重组甘精胰岛素原cDNA片段插入测序质粒pUC18中,命名为pUC18-ProINsa转化大肠杆菌DH5a,进行测序。划线接种在LA(含100ug/ml氨苄青霉素的LB)琼脂平板上,37℃过夜培养进行活化。挑取单克隆接种于含100ug/ml氨苄青霉素的LB培养基中(即LA培养基),37℃,200rpm过夜培养。以20%甘油和LA培养基保存菌种。按照小剂量质粒提取试剂盒提供的方法提取质粒pUC18-ProINsa。因质粒pUC18多克隆位点上游第185位有Nde I识别位点,为在琼脂糖凝胶中更明显地区分,先用BamHI和Sac I双酶切pUC18-ProINsa,1%的低溶点琼脂糖凝胶回收320bp左右的小片段,再用Nde I单切小片段并回收310bp左右的片段待连接用。
将质粒pET-3c用Nde I和BamHI进行双酶切,1%的低溶点琼脂糖凝胶回收大片段,与上述已经回收的310bp左右的片段在T4连结酶的作用下4℃过夜连结。氯化钙法转化大肠杆菌DH5α,平铺LA琼脂平板,37℃过夜培养。次日挑取8个单菌落进行摇瓶培养,从6个菌落中提取到质粒。接着对质粒进行了BamH I、Nde I、Xba I单酶切和BamH I/Nde I、BamH I/Xba I双酶切的鉴定。结果显示,经BamH I、Nde I、Xba I单酶切后,2%琼脂糖电泳可见所提质粒单酶切后呈线性。经BamH I/Nde I、BamH I/Xba I双酶切后,1%琼脂糖电泳可见质粒被酶切开,形成4300bp左右和310bp左右两个线性片段。鉴定正确后的质粒即为重组甘精胰岛素原的重组表达质粒,命名为pET-ProINsa。
二、重组甘精胰岛素原表达质粒的筛选和鉴别
将鉴定后的pET-ProINsa质粒用氯化钙法转化大肠杆菌宿主菌BL21(DE3)PlysS,从中随机选10个克隆进行表达。即用单克隆用牙签接种于含100ug/ml氨苄青霉素的LA培养基中,37℃,180rpm过夜。按1∶10接种于LA培养基中,37℃,250rpm,测培养基OD600值为0.6时,加入IPTG使终浓度为0.5mM继续同样条件诱导4小时,取样进行15%的SDS-PAGE电泳。其目的蛋重组甘精胰岛素类似物应在10KD分子量处。实验结果提示每个克隆菌中均有目的蛋白表达,其中4个克隆菌表达最高,目的蛋白表达量达40%左右。保存菌株,命名为pET-ProINsa/BL21(DE3)PlysS。
保存的菌种进行LA斜面活化后,再从活性斜面上进行种子培养(37℃,180rpm,LA培养基)。按1∶10接种1000ml LA培养基中,37℃,250rpm,培养基OD600值0.6左右时加入0.2mM IPTG诱导(37℃,250rpm)1,2,3,4小时,分别取样,进行15%SDS-PAGE电泳,结果显示3小时其表达量已达到最高。
以1g菌体与10ml破菌缓冲液(50mM Tris-Hcl pH 9.0、5mM EDTA、1%Triton-X100、2M尿素)的比例悬浮菌体,室温震荡混匀,经超声处理后离心收集上清和沉淀,沉淀用8M尿素溶解。上清和沉淀均进行15%SDS-PAGE电泳,结果显示表达的目的蛋白95%以上在沉淀中,证明重组甘精胰岛素原主要以包涵体形式表达。
实例3.大肠杆菌表达含新型C肽重组甘精胰岛素原纯化
和活性重组甘精胰岛素制备
一、重组甘精胰岛素原的发酵
取-70℃保存菌种划线接种于LA(1%Trypton,0.5%Yeast extract,1%NaCl,AMP100ug/ml)斜面,置37℃恒温孵箱15hr进行pET-ProINsa/BL21(DE3)PlysS的活化。挑取单克隆菌落接种到50ml LA中37℃,150rpm培养8-10hr,以1∶10接种1000mlLA中37℃,150rpm培养12-14hr,加入9L M9YT(M9YT 10L体系:Na2HPO4·7H2O1758,KH2PO4 30g,NH4Cl 10g,NaCl 5g,Trypton 50g,Yeast extract 30g,MgSO4·7H2O10g)培养基中。调节发酵启始参数为:300rpm、37℃、pH7.0、DO100%、通气量1vvm。50%氨水自动维持pH7.0,通过提高转速和提高氧气维持DO≥30%。葡萄糖接近被消耗完时开始流加营养液,维持葡萄糖终浓度0.3%至OD600=15,停止补料。DO曲线上升至约50-60%,加入终浓度0.3mM IPTG诱导,维持葡萄糖终浓度为0.3%至结束。诱导3小时后发酵结束。4℃、7000rpm离心15min收集菌体,-20℃保存。
二、重组甘精胰岛素原的复性和纯化
以1G菌体:10ml破菌缓冲液(50mM Tris-Hcl PH 9.0、5mM EDTA、1%Triton-X100、2M尿素)悬浮菌体,待完全溶解,经高压匀质机30-40Mpa/2-3次破菌,12000rpm/15min离心收集沉淀。用25mM Tris-HCl pH 9.0、5mM EDTA、1M NaCl、2M尿素搅拌洗涤包涵体30min,12000rpm/15min离心收集沉淀。将沉淀用50mM Tris-Hcl pH 10.5、5mMEDTA、1/1000 β-Me、8M Urea溶解,搅拌洗涤30min,12000rpm/15min离心收集上清。以25mMTris-HCl pH 10.5、0.5g/L L-Cysteine hydrochloride复性液逐步稀释复性(4℃中,100rpm搅拌下),经3KD超滤浓缩20倍。
10mM乙酸-乙酸钠pH3.0平衡S.P.Seplarose FF层析柱,乙酸调样品pH 3.0后上样,10mM乙酸-乙酸钠pH6.0洗脱杂峰,50mM Tris pH 10.0洗脱目的峰。S.P.SepharoseFF层析柱洗脱峰加入终浓度0.3M NaCl,调pH9.0,上样25mM Tris pH 9.0和0.3M NaCl平衡后的Ni+层析柱(Ni-Chelating Sepharose),25mM Tris pH 9.0/0.5M NaCl/50mM咪唑洗脱杂峰,25mM TrisPH 9.0/200mM咪唑洗脱重组甘精胰岛素原。
三、重组甘精胰岛素的制备
重组甘精胰岛素原蛋白用1M HCl调pH7.0沉淀,离心后用25mM Tris pH 9.0溶解,调成10mg/ml蛋白浓度。按1∶1000(质量比)加入胰蛋白酶(TPCK处理,Sigma产品),25℃水浴,50rpm共1小时,1M HCl调pH 3.0终止反应。酶切后样品加入缓冲液A(5%乙氰/0.1%TFA)平衡的C18反向层析柱,35%缓冲液B(70%乙氰/0.2%TFA)洗脱杂质峰,35%-45%缓冲液B梯度洗脱重组甘精胰岛素。抽滤除去乙氰后调成含50mM Tris pH 9.0的溶液,上样相同缓冲体系平衡的Ni+层析柱。收集Ni+层析柱穿透峰,1M HCl调pH7.0,10000rpm离心30分钟收集沉淀。用pH4.0,10mM乙酸-乙酸钠缓冲溶解沉淀,即得精制得重组甘精胰岛素。
四、重组甘精胰岛素高效液相(HPLC)分析
方法:采用C18高效液相色谱柱(4.6mm×250mm),流动相A:磷酸盐缓冲液(pH2.5)∶乙腈∶水∶氯化钠=250ml∶250ml∶400ml∶18.4g,加水至1000ml,0.45um滤膜过滤。流动相B:磷酸盐缓冲液(pH2.5)∶乙腈∶水∶氯化钠=250ml∶650ml∶50ml∶3.2g,加水至1000ml,0.45um滤膜过滤。流速1.0ml/min。洗脱梯度:4%B 0-20min,17%B 20-30min,37%B 30-40min,4%B 40min。柱温35℃。检测波长214nm。
结果:经纯化所得重组甘精胰岛素纯度大于99%。
实例4.大肠杆菌表达重组甘精胰岛素的延缓降糖作用
一、材料和方法
试验动物:雄性家兔18只,2.0-3.0kg/只。
受试药物:重组甘精胰岛素,3.64mg/ml/支。加入溶剂使成10ml,每支含30mcg锌离子、2.7mg甲酚、20mg 85%甘油,盐酸调节pH值到4.0左右。用生理盐水将胰岛素配制成1.2IU/ml,4-8℃保存不超过5天。
对照药物:重组甘精胰岛素(商品名为Lantus,Aventis公司),规格为100IU/3.6378mg/10ml/支。每支含30mcg锌离子、2.7mg甲酚、20mg 85%甘油,盐酸调节pH值到4.0左右。用生理盐水将胰岛素配制成1.2U/ml,用前配制。
国家标准品:国家标准胰岛素,264IU/10.0mg//支,用受试药物和对照药物的配方配制成1.2IU/ml。用生理盐水将胰岛素配制成0.06-0.12U/ml。用前配制。
方法:将家兔分为受试药物组、对照药物组和国家标准品组共3组,每组6只。给药前分别自右侧耳缘静脉取血1.0ml,检测血糖浓度。2小时之后家兔单次左侧耳缘静脉注射配制好的受试药物、对照药物和国家标准品各1.0ml。国家标准品组在给药后2小时和6小时,受试药物组在给药后6小时和9小时,再分别自右侧耳缘静脉取血1.0ml,检测血糖浓度。
二、结果
所有家兔未发生痉挛和死亡。各试验组用药后降低血糖浓度降低的比例见下表:
各试验组用药后2小时降低血糖浓度的比例(%)
试验组 | 家兔编号 | 平均 | |||||
1号 | 2号 | 3号 | 4号 | 5号 | 6号 | ||
受试药物组(6hr)受试药物组(9hr)对照药物组(6hr)对照药物组(9hr)国家标准品组(2hr)国家标准品组(6hr) | 717068696196 | 646573725798 | 686963646399 | 636474735697 | 717171716498 | 676866646196 | 66.8367.8369.1768.8360.3397.33 |
证明本专利方法制备的重组甘精胰岛素与国外同类产品的降血糖生物作用一致,其生物比活性经国家标准品胰岛素测定为30IU/mg。
附图说明
图1中的“Ni+层析(Ni-Chelating Sepharose)纯化甘精胰岛素原前后的非还原SDS-PAGE凝胶电泳图”
图2中的“纯化后所得重组甘精胰岛素经反相高效液相(HPLC)分析结果”以及“分析结果表:
分析结果表
峰号 峰名 保留时间 峰高 峰面积 含量
1 9.490 520694.250 11458496.000 99.6048
2 11.892 1201.533 38462.777 0.3343
3 12.620 349.015 6998.600 0.0608
总计 522244.799 11503957.377 100.0000
经Ni+鏊合层析后,C18反向HPLC分析重组甘精胰岛素纯度大于99%。”
电子可读序列-glargine专利
高效表达重组人胰岛素原及其类似物的新型C肽序列表
Organization Applicant
----------------------
Street:重庆市石桥铺科园四街70号
City:重庆市
State:重庆市
Country:中华人民共和国
PostalCode:400041
PhoneNumber:023-68888852
FaxNumber:023-68699676
EmailAddress:fankai1963@yahoo.com.cn
<110>OrganizationName:重庆富进生物医药有限公司
Application Project
-------------------
<120>Title:高效表达重组人胰岛素原及其类似物的新型C肽
<130>AppFileReference:
<140>CurrentAppNumber:
<141>CurrentFilingDate:
<160>4
<170>PatentIn version 3.2
Sequence:Seq1
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
Met Ala Thr Thr Ser Thr Ala Thr Thr Arg 10
<212>Type:PRT
<211>Length:10
SequenceName:重组人胰岛素原引导肽氨基酸序列
Feature
-------
<221>FeatureKey:mat_peptide
<222>LocationFrom:1
<222>LocationTo:10
Other Information:重组人胰岛素原引导肽氨基酸序列
CDS Join:Yes
Sequence:Seq2
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu 15
Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr 30
Arg Arg 32
<212>Type:PRT
<211>Length:32
SequenceName:重组甘精胰岛素B链氨基酸序列
Feature
-------
<221>FeatureKey:mat_peptide
<222>LocationFrom:1
<222>LocationTo:32
Other Information:重组甘精胰岛素B链氨基酸序列
CDS Join:No
Sequence:Seq3
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
Glu Val Pro His His His His His Gln Val Glu Leu Gly Gly Gly 15
Pro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu 30
Gln Lys Arg 33
<212>Type:PRT
<211>Length:33
电子可读序列-glargine专利
SequenceName:重组甘精胰岛素原新型C肽氨基酸序列
Feature
-------
<221>FeatureKey:mat_peptide
<222>LocationFrom:1
<222>LocationTo:33
Other Information:重组甘精胰岛素原新型C肽氨基酸序列
CDS Join:No
Sequence:Seq4
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln 15
Leu Glu Asn Tyr Cys G1y 21
<212>Type:PRT
<211>Length:21
SequenceName:重组甘精胰岛素A链氨基酸序列
Feature
-------
<221>FeatureKey:mat_peptide
<222>LocationFrom:1
<222>LocationTo:21
Other Information:重组甘精胰岛素A链氨基酸序列
CDS Join:No
Sequence:Seq5
--------
<213>OrganismName:人工序列
<400>PreSequenceString:
CTAATGGCTA CAACCTCTAC AGCTACCACA CGTTTCGTTA ACCAGCACCT GTGCGGTTCT 60
CACCTGGTTG AAGCTCTGTA CCTGGTTTGC GGTGAACGTG GTTTCTTCTA CACCCCGAAA 120
ACCCGTCGTG AAGTTCCGCA CCATCACCAT CACCAGGTTG AACTGGGTGG TGGTCCGGGT 180
GCTGGTTCTC TGCAGCCGCT GGCTCTGGAA GGTTCTCTGC AGAAACGTGG TATCGTTGAA 240
CAGTGCTGCA CCTCTATCTG CTCTCTGTAC CAGCTGGAAA ACTACTGCGG TTAAGGATCC 300
<212>Type:DNA
<211>Length:300
SequenceName:用于原核细胞表达质粒构建的重组甘精胰岛素原cDNA序列
Feature
-------
<221>FeatureKey:cDNA
<222>LocationFrom:1
<222>LocationTo:300
Other Information:用于原核细胞表达质粒构建的重组甘精胰岛素原cDNA序列
CDS Join:No
Claims (10)
1、本专利涉及一种用重组制备胰岛素原及其类似物的新型C形肽。
2、条款1中所述新型C肽的特征为人胰岛素原C肽的N末端4-12位氨基酸经4-8个组氨酸替换而成。其序列表现形式为Glu-His(4-8)-C肽。
3、条款2中所述新型C肽的第1位氨基酸为Glu。第2-10位氨基酸用4-8个His串联替换人胰岛素原相对应的氨基酸,同时包括任意替换相对应的氨基酸,未替换的氨基酸同人胰岛素C肽的氨基酸序列。
4、条款2中所述新型C肽替换组氨酸后由33个氨基酸组成。
5、条款1中所述新型C肽适用于重组人胰岛素原的高效表达。
6、条款1中所述新型C肽适用于重组人胰岛素原类似物的高效表达。
7、条款6中的重组人胰岛素原类似物包括甘精胰岛素、三精胰岛素、谷甘胰岛素、赖脯胰岛素、精蛋白锌胰岛素等。
8、含新型C肽重组表达的人胰岛素原及其类似物用金属离子鏊和层析纯化。
9、经胰蛋白酶或/和羧肽酶B酶切后的新型C肽用金属离子鏊和层析去除。
10、本专利所述新型C肽适用于原核及真核表达体系表达重组人胰岛素原及其类似物。
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US5514646A (en) * | 1989-02-09 | 1996-05-07 | Chance; Ronald E. | Insulin analogs modified at position 29 of the B chain |
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