CN110564797A - 一种利用热稳定融合蛋白制备多肽的方法 - Google Patents

一种利用热稳定融合蛋白制备多肽的方法 Download PDF

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CN110564797A
CN110564797A CN201910751657.4A CN201910751657A CN110564797A CN 110564797 A CN110564797 A CN 110564797A CN 201910751657 A CN201910751657 A CN 201910751657A CN 110564797 A CN110564797 A CN 110564797A
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刘懿
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Abstract

本发明涉及一种基因重组大肠杆菌表达的融合蛋白制备方法，属于生物工程技术领域。针对目前各种大肠杆菌工程菌表达的融合蛋白制备工艺中的缺点，特别是菌体破胞这一步的难点，开发了一种适合工业化大生产的解决方案。其主要特征在于，发酵结束后，在不需要离心收集菌体的前提下，利用发酵液直接高温热破胞的方式达到高效的菌体破胞效果，再经过碟片离心机离心获得含有重组融合蛋白的上清液。然后利用膜过滤和膜浓缩常规流程获得浓缩后的重组融合蛋白溶液，然后经酶切获得目标多肽。该工艺设备要求低，工艺流程简单、成本低，非常适合工业化放大生产。

Description

一种利用热稳定融合蛋白制备多肽的方法
技术领域
本发明属于发酵工程领域，尤其涉及一种利用热稳定融合蛋白制备多肽的方法。
背景技术
利用大肠杆菌系统生产蛋白或多肽时，在提取过程中往往需要将菌体细胞破碎，把目标多肽释放出来，然后进一步提取。目前破胞的方法较多，比如超声法、高压均质、有机溶剂法、酶法、冻融法等，这些方法各自都存在一定的局限性，超声法破胞能耗较高不适于工业化放大生产，冻融法破胞需要的时间较长，高压均质破胞产生的热量较高容易对酶造成伤害，有机溶剂法缺点也非常明显，添加的有机溶剂会带来额外的环保压力，同时有机溶剂还可能对目标多肽带来伤害，而法破胞用的酶，破胞后释放出来的蛋白有可能会被其降解。现存的破胞方法在成本上或者规模上都不能达到最好的效果，所以急需寻找一种成本低又易于工业化生产的破胞提取方法。
发明内容
为了解决上述问题，本发明提供了一种针对热稳定性蛋白的有效制备提取工艺。
本发明的技术方案包括：
一种利用热稳定融合蛋白制备多肽的方法，它包括如下步骤：
1)使用重组大肠杆菌发酵表达融合蛋白，该融合蛋白是热稳定蛋白与目标多肽的融合蛋白；
2)将破胞缓冲液加热到60～100℃，与发酵液按质量比1∶(0.5～4)混合，同时加入相当于混合液质量0.01％-2％的表面活性剂，搅拌破胞；
3)离心取上清；
4)过滤除杂，超滤浓缩；
5)酶切，得目标多肽；
步骤1)所述热稳定蛋白是在60～100℃高温下维持稳定的蛋白，所述融合蛋白也能在60～100℃高温下维持稳定；
步骤2)中，表面活性剂是非离子型表面活性剂；
步骤2)中，所述破胞缓冲液能使破胞后混合溶液的pH值与融合蛋白等电点的差值的绝对值在2以上。
优选地，步骤2)搅拌时间是5～60min。
如前述的方法，其特征在于：
步骤2)中的表面活性剂是聚醚类、聚乙二醇类、酯类或多元醇类非离子型表面活性剂，优选地，为曲拉通X-100或吐温-80。
如前述的方法，其特征在于：
步骤2)中的盐类缓冲液，所述的盐类选自磷酸盐、Tris-HCl、乙酸盐；和/或，浓度范围是10-100mM。
如前述的方法，其特征在于：
步骤3)所述离心为连续离心，优选地，为碟片式离心。
如前述的方法，其特征在于：
步骤3)中离心后的上清液中不溶物湿重不超过上清液的3％，优选地，不超过上清液的1％。
如前述的方法，其特征在于：
步骤4)中，过滤除杂使用PP材质的中空纤维膜或PES材质的微滤膜；和/或，超滤浓缩采用的膜是PES材质的。
如前述的方法，其特征在于：
步骤4)的超滤过程中，料液温度不超过40℃；和/或，压力不超过0.4MPa；和/或，pH值不超过8.5；
优选地，步骤4)的超滤过程中，料液温度不超过35℃；和/或，压力补偿不超过0.2MPa；和/或，pH值不超过7.8。
如前述的方法，其特征在于：
步骤5)中的酶是肠激酶、胰蛋白酶或赖氨酰肽链内切酶，优选地，为肠激酶。
如前述的方法，其特征在于：
步骤5)中反应在水相或者较低比例有机相体系中进行；
所述有机相是乙腈或者短链醇类；所述短链指的是分子中碳原子个数不超过3；
所述较低比例指比例不超过25％，优选地不超过10％，最优选地是低于5％。
如前述的方法，
步骤1)所述热稳定蛋白序列如SEQ ID NO.1～3中任一所示；
和/或，目标多肽的序列如SEQ ID NO.36～43中任一所示；
和/或，融合蛋白中，热稳定蛋白与目标多肽之间还有一段连接肽。
优选地，融合蛋白的序列如SEQ ID NO.4～35任一所示。
如前述的方法，融合蛋白的序列如SEQ ID NO.26所示。
本发明具有如下有益效果：
本发明的制备多肽的方法十分便利，设备要求低，破胞快速，成本低，所得蛋白收率高，活性高，非常适合大规模生产。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过具体实施方式对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1：高温破胞后的电泳图
图2：融合蛋白(SEQ ID NO.4)生产多肽P1结果HPLC图
图3：融合蛋白(SEQ ID NO.5)生产多肽P1结果HPLC图
图4：融合蛋白(SEQ ID NO.6)生产多肽P1结果HPLC图
图5：融合蛋白(SEQ ID NO.7)生产多肽P1结果HPLC图
图6：融合蛋白(SEQ ID NO.10)生产多肽P2结果HPLC图
图7：融合蛋白(SEQ ID NO.11)生产多肽P2结果HPLC图
图8：融合蛋白(SEQ ID NO.16)生产多肽P3结果HPLC图
图9：融合蛋白(SEQ ID NO.17)生产多肽P3结果HPLC图
图10：融合蛋白(SEQ ID NO.22)生产多肽P3结果HPLC图
图11：融合蛋白(SEQ ID NO.23)生产多肽P4结果HPLC图
图12：融合蛋白(SEQ ID NO.24)生产多肽P5结果HPLC图
图13：融合蛋白(SEQ ID NO.25)生产多肽P5结果HPLC图
图14：融合蛋白(SEQ ID NO.26)生产多肽P5结果HPLC图
图15：融合蛋白(SEQ ID NO.27)生产多肽P6结果HPLC图
图16：融合蛋白(SEQ ID NO.30)生产多肽P7结果HPLC图
图17：融合蛋白(SEQ ID NO.33)生产多肽P8结果HPLC图
具体实施方式
本发明大体涉及如下的步骤：
1、重组大肠杆菌的发酵；
2、重组大肠杆菌的高温破胞；
3、离心分离；
4、膜分离及膜浓缩；
5、酶切。
本发明具体的多肽制备方法包括：
一级种培养条件：接种量：0.1％；培养温度37℃，摇床转速220rpm，培养7-9h，至OD600为3.0-7.0。
所述一级种培养基组成：
原料 成分配比
胰蛋白胨 1％
酵母提取物 0.5％
氯化钠 1％
二级种培养条件：接种量：0.1％；培养温度34℃，摇床转速220rpm，培养10-12h，至OD600为2.0-4.0。
所述二级种培养基组成：
原料 成分配比
磷酸氢二铵 0.200％
磷酸二氢钾 0.675％
一水柠檬酸 0.093％
七水硫酸镁 0.070％
Trace solution 0.25％
一水葡萄糖 2％
pH 6.8
所述罐发酵培养基组成：
所述培养基中所涉及的微量元素溶液(Trace solution)组成为：
成分 含量
FeSO4·7H2O 10g/L
MnSO4·5H2O 0.5g/L
ZnSO4·7H2O 2.25g/L
CuSO4·5H2O 1g/L
CaCl2·2H2O 2g/L
Na2B4O7·10H2O 0.23g/L
(NH4)6Mo7O24 0.1g/L
HCl 5mol/L
罐发酵将二级种子培养液按照2-5％的接种量接种入发酵罐，然后按照以下参数进行第一阶段罐发酵(初始生长阶段)：
温度：37℃；
pH：6.8±0.2；
转速：初始200-250rpm；当溶氧低于30％时，转速增加至700-1000rpm。
通气：初始1：0.5vvm；
罐压：0.05MPa；
溶氧：大于或等于20％；
发酵起始溶氧大于90％，溶氧随着菌体生长逐渐降低，当溶氧飙升至80％时进入第二阶段发酵(分批补料阶段)，第二阶段发酵按照如下参数调控：
温度：35℃；
pH：6.8±0.2；
转速：750-1000rpm；
通气：初始1：1.5vvm；
罐压：0.05MPa
溶氧：大于或等于20％；
补料时间：6.0±1.0h；
补料成分：70％的一水葡萄糖，2％的七水硫酸镁，0.005％的硫酸卡那霉素。
当发酵液湿重(指发酵液中不溶物的湿重)达到160±20g/kg时，进入第三阶段发酵(蛋白诱导阶段)，补加诱导剂，按照以下参数调控：
温度：30℃；
pH：6.8±0.2；
转速：800-1000rpm；
通气：初始1：2vvm；
罐压：0.05MPa；
溶氧：大于30％；
诱导剂：40％乳糖；
诱导剂工作浓度：乳糖工作浓度为初始工作体积的2％；
诱导时间：11±1h
具体方法还包括如下的提取步骤：
(1)高温破胞步骤：
将破胞缓冲液加热到60-100℃，按照破胞缓冲液与发酵液的质量比为1∶0.5-1∶4的量向其中加入发酵液，同时加入0.01％-2％的表面活性剂，搅拌破胞，破胞时间为5-60min。
所述的破胞缓冲液，其组成为盐种类包括磷酸盐、Tris-HCl、乙酸盐等。浓度范围为10-100mM；且破胞缓冲液能使破胞后混合溶液的pH值与融合蛋白等电点的差值的绝对值在2以上。
所述表面活性剂其种类包括聚醚类、聚乙二醇类、酯类及多元醇类等非离子型表面活性剂。其中较优的选择包括曲拉通X-100，吐温80等。
(2)离心步骤：采用连续离心的方式，特别优选的是碟片式离心，其中离心后的上清液湿重(指上清液中不溶物湿重)不超过3％，优选的不超过1％。
(3)过滤和浓缩步骤：采用错流式膜过滤系统获得澄清料液，再采用超滤膜来浓缩料液。过滤膜采用PP材质的中空纤维膜或PES材质的微滤膜，超滤膜采用PES材质的。超滤膜操作过程中，维持料液温度不超过40℃，压力不超过0.4MPa。优选的温度不超过35℃，压力不超过0.2MPa；操作过程中维持料液的pH值不超过8.5，优选的维持pH不超过7.8。
(4)酶切步骤：该步骤可采用肠激酶、胰蛋白酶、赖氨酰肽链内切酶等，工艺中较优的选择是肠激酶。反应在水相或者含较低比例有机相体系中进行，所含有机相包括甲醇、乙醇等短链醇类。有机相比例不超过25％，优选的不超过10％，最优的选择是低于5％。
本方法所述的蛋白主要包括但不限于具有如下氨基酸序列的蛋白：
本方法所述的目的多肽主要包括但不限于具有如下氨基酸序列的多肽。
下述实施例中所用的实验材料，在没有特殊说明的情况下均为自常规生化试剂厂商处购得。
实施例1、重组大肠杆菌菌发酵表达融合蛋白mTrA-Arg34-GLP-1(7-37)(SEQ IDNO.4)
将制备好的重组大肠杆菌(E.coli BL21(DE3)/pET28a-mTrA-Arg34-GLP-1(7-37))二级种子培养液2000ml种入100L发酵罐(装量50L)，同时加入26ml卡那霉素溶液，后按照以下参数进行第一阶段罐发酵：温度：37℃；pH：6.8；转速：初始250rpm；当溶氧低于30％时，转速逐渐提高至750rpm；通气：初始1：0.5vvm(air volume/culture volume/min，通气比)；溶氧：大于或等于20％；
当溶氧飙升至80％时进入第二阶段发酵，第二阶段发酵按照如下参数调控：温度：35℃；pH：6.8；转速：750rpm；通气：初始1：1.5vvm；溶氧：大于或等于20％；补料(持续)时间：5.5h；补料培养基：70％(kg/L)一水葡萄糖，2％(kg/L)七水硫酸镁，0.005％(kg/L)卡那霉素；补料用培养基的体积为7～8L，优选7.5L。
当发酵液湿重高于165g/kg时，开始第三阶段发酵，补加诱导剂，按照以下参数调控：温度：30℃；pH：6.8；转速：800rpm；通气：初始1：2vvm；溶氧：大于或等于20％；诱导剂：40％乳糖；诱导剂工作浓度：乳糖工作浓度为初始工作体积的2％；诱导时间达到11h时，发酵结束。所得发酵液重量为78.3kg。
实施例2、重组融合蛋白mTrA-Arg34-GLP-1(7-37)(SEQ ID NO.4)的提取
配制40mM的磷酸盐缓冲溶液156kg，用氢氧化钠溶液调pH至9.5，得破胞缓冲液，加入发酵罐中，通过夹套蒸汽加热，同时以100rpm的转速搅拌，使溶液温度上升至80℃以上，将实施例1中所得发酵液78.3kg加入破胞缓冲液中，同时向其中加入500g曲拉通X-100；维持搅拌待破胞完成，开始离心。将清洗干净的碟片离心机开机，通过调节进料速度、排渣周期及排渣时间等参数将离心后上清液的湿重控制在2％以内，将离心上清液温度降至25℃，调节上清液pH值为7.5。上清液通过中空纤维膜过滤，中空纤维膜的材质为聚丙烯树脂材质。控制操作压力不超过0.04MPa，用pH7.5的20mM的磷酸盐buffer顶洗截留液，合并所得滤过液。再通过超滤膜进行浓缩。超滤膜的截留分子量为10万道尔顿，超滤膜的材质为PES材质聚醚砜。控制过程中压力不超过0.2MPa，温度不超过30℃，浓缩后再用30kg的20mM的Tris或磷酸盐buffer(pH7.5)顶洗，顶洗完后终体积为15kg。
实施例3、重组大肠杆菌菌发酵表达融合蛋白PTH1-N(SEQ ID NO.26)
按照实施例1的条件进行SEQ ID NO.26重组蛋白的发酵，最终获得发酵液78.5kg。
发酵液按实施例2的方法进行破胞和离心，将得到的上清液和沉淀用蛋白电泳检测，结果发现，上清液中出现较为纯净的融合蛋白PTH1-N(SEQ ID NO.26)的条带，而沉淀中等分子量的条带颜色浅很多(图1)，表明本实施例的PTH1-N蛋白产物绝大多数在离心上清中。
实施例4、融合蛋白PTH1-N(SEQ ID NO.26)的提取
配制40mM的磷酸盐缓冲溶液155kg，用氢氧化钠溶液调pH至9.5，得破胞缓冲液，加入发酵罐中，通过夹套蒸汽加热，同时以100rpm的转速搅拌，使溶液温度上升至85℃以上，将实施例3中所得发酵液78.5kg加入破胞缓冲液中，同时向其中加入480g曲拉通X-100；维持搅拌至破胞完成，开始离心。将清洗干净的碟片离心机开机，通过调节进料速度、排渣周期及排渣时间等参数将离心后上清液的湿重控制在2％以内，将离心上清液温度降至25℃，调节上清液pH值为7.5。上清液通过中空纤维膜过滤，中空纤维膜的材质为聚丙烯树脂材质。控制操作压力不超过0.04MPa，用pH7.5的20mM的磷酸盐buffer顶洗截留液，合并所得滤过液。再通过超滤膜进行浓缩。超滤膜的截留分子量为10万道尔顿，超滤膜的材质为PES材质聚醚砜。控制过程中压力不超过0.2MPa，温度不超过30℃，浓缩后再用20mM的Tris或磷酸盐buffer(pH7.5)30kg顶洗，顶洗完后终体积为15.6kg。
实施例5、重组大肠杆菌菌发酵表达融合蛋白mTrA-Arg34-GLP-1(9-37)(SEQ IDNO.10)
将产氨基酸序列为SEQ ID NO.10融合蛋白的基因工程菌的二级种子培养液500mL(3.57％(mL/mL))接种入30L发酵罐(装量14L)，同时加入7mL硫酸卡那霉素溶液，后按照以下参数进行第一阶段罐发酵：温度：37℃；pH：6.8；转速：初始250rpm；当溶氧低于30％时，转速逐渐提高至750rpm；通气：初始1：0.5vvm；溶氧：大于或等于20％；
当溶氧飙升至80％时开始第二阶段发酵，第二阶段发酵按照如下参数调控：温度：35℃；pH：6.8；转速：750rpm；通气：初始1：1.5vvm；溶氧：大于等于20％；补料时间：5.5h；补料成分：70％(Kg/L)一水葡萄糖，2％(Kg/L)七水硫酸镁，0.005％(Kg/L)硫酸卡那霉素。
当发酵液湿重大于150g/kg时，开始第三阶段发酵，补加诱导剂，按照以下参数调控：温度：30℃；pH：6.8；转速：800rpm；通气：初始1：2；溶氧：大于30％；诱导剂：40％乳糖；诱导剂工作浓度：乳糖工作浓度为初始工作体积的2％；诱导时间达到11h时，发酵结束。所得发酵液总量为38.5kg。
实施例6、融合蛋白mTrA-Arg34-GLP-1(9-37)(SEQ ID NO.10)的提取
配制40mM的磷酸盐缓冲溶液78kg，用氢氧化钠溶液调pH至9.5，得破胞缓冲液，加入发酵罐中，通过夹套蒸汽加热，同时以100rpm的转速搅拌，使溶液温度上升至80℃以上，将实施例5中所得发酵液38.5kg加入破胞缓冲液中，同时向其中加入250g曲拉通X-100；维持搅拌至破胞完成，开始离心。将清洗干净的碟片离心机开机，通过调节进料速度、排渣周期及排渣时间等参数将离心后上清液的湿重控制在2％以内，将离心上清液温度降至25℃，调节上清液pH值为不超过7.5。上清液通过中空纤维膜过滤，中空纤维膜的材质为聚丙烯树脂材质。控制操作压力不超过0.04MPa，用pH7.5的20mM的磷酸盐buffer顶洗截留液，合并所得滤过液。再通过超滤膜进行浓缩。超滤膜的截留分子量为10万道尔顿，超滤膜的材质为PES材质聚醚砜。控制过程中压力不超过0.2MPa，温度不超过30℃，浓缩后再用30kg 20mM的Tris或磷酸盐buffer(pH7.5)顶洗，顶洗完后终体积为14.16kg。
实施例7、重组大肠杆菌菌发酵表达融合蛋白mTrA-Arg34-GLP-1(11-37)(SEQ IDNO.16)
按照实施例5的条件进行SEQ ID NO.16融合蛋白的发酵，最终获得发酵液38.1kg。
实施例8、融合蛋白mTrA-Arg34-GLP-1(11-37)(SEQ ID NO.16)的提取
配制40mM的磷酸盐缓冲溶液78kg，用氢氧化钠溶液调pH至9.5，得破胞缓冲液，加入发酵罐中，通过夹套蒸汽加热，同时以100rpm的转速搅拌，使溶液温度上升至80℃以上，将实施例7中所得发酵液38.1kg加入破胞缓冲液中，同时向其中加入250g曲拉通X-100；维持搅拌至后破胞完成，开始离心。将清洗干净的碟片离心机开机，通过调节进料速度、排渣周期及排渣时间等参数将离心后上清液的湿重控制在2％以内，将离心上清液温度降至25℃，调节上清液pH值为不超过7.5。上清液通过中空纤维膜过滤，中空纤维膜的材质为聚丙烯树脂材质。控制操作压力不超过0.04MPa，用pH7.5的20mM的磷酸盐buffer顶洗截留液，合并所得滤过液。再通过超滤膜进行浓缩。超滤膜的截留分子量为10万道尔顿，超滤膜的材质为PES材质聚醚砜。控制过程中压力不超过0.2MPa，温度不超过30℃，浓缩后再用20mM的Tris或磷酸盐buffer(pH7.5)30kg顶洗，顶洗完后终体积为15.3kg。
实施例9、重组大肠杆菌菌发酵表达融合蛋白GLP-2-N(SEQ ID NO.22)
按照实施例1的条件进行SEQ ID NO.22融合蛋白的发酵，最终获得发酵液77.4kg。
实施例10、融合蛋白GLP-2-N(SEQ ID NO.22)的提取
配制40mM的磷酸盐缓冲溶液158kg，用氢氧化钠溶液调pH至9.5，得破胞缓冲液，加入发酵罐中，通过夹套蒸汽加热，同时以100rpm的转速搅拌，使溶液温度上升至85℃以上，将实施例9中所得发酵液77.4kg加入破胞缓冲液中，同时向其中加入400g曲拉通X-100；维持搅拌至破胞完成，开始离心。将清洗干净的碟片离心机开机，通过调节进料速度、排渣周期及排渣时间等参数将离心后上清液的湿重控制在2％以内，将离心上清液温度降至25℃，调节上清液pH值为7.5。上清液通过中空纤维膜过滤，中空纤维膜的材质为聚丙烯树脂材质。控制操作压力不超过0.04MPa，用pH7.5的20mM的磷酸盐buffer顶洗截留液，合并所得滤过液。再通过超滤膜进行浓缩。超滤膜的截留分子量为10万道尔顿，超滤膜的材质为PES材质聚醚砜。控制过程中压力不超过0.2MPa，温度不超过30℃，浓缩后再用20mM的Tris或磷酸盐buffer(pH7.5)30kg顶洗，顶洗完后终体积为14.8kg。
实施例11、重组大肠杆菌菌发酵表达融合蛋白PTH2-1(SEQ ID NO.27)
按照实施例5的条件进行SEQ ID NO.22融合蛋白的发酵，最终获得发酵液38kg。
实施例12、融合蛋白PTH2-1(SEQ ID NO.27)提取
按照实施例6的条件进行SEQ ID NO.27融合蛋白的提取，最终获得浓缩融合蛋白溶液7kg。
实施例13、重组大肠杆菌菌发酵表达融合蛋白PTH3.1(SEQ ID NO.30)
按照实施例5的条件进行SEQ ID NO.30融合蛋白的发酵，最终获得发酵液38.2kg。
实施例14、融合蛋白PTH3-1(SEQ ID NO.30)提取
按照实施例6的条件进行SEQ ID NO.30融合蛋白的提取，最终获得浓缩融合蛋白溶液7.4kg。
实施例15、重组大肠杆菌菌发酵表达融合蛋白GC-1(SEQ ID NO.33)
按照实施例5的条件进行SEQ ID NO.33融合蛋白的发酵，最终获得发酵液38.8kg。
实施例16、融合蛋白GC-1(SEQ ID NO.33)提取
按照实施例6的条件进行SEQ ID NO.33融合蛋白的提取，最终获得浓缩融合蛋白溶液7.5kg。
实施例17、重组融合蛋白mTrA-Arg34-GLP-1(7-37)(SEQ ID NO.4)的酶切
将实施例2所得的15kg融合蛋白溶液经0.22μm的滤膜过滤后加入灭菌后的30L的反应釜中，调节pH至8.5，维持温度22℃左右，加入肠激酶液50ml(3000u/ml)，缓慢搅拌反应，HPLC监控反应进度，8h后反应结束。用2N盐酸调体系pH至4.7，降温至4-8℃，静置4h，离心获得目的多肽Arg34-GLP-1(7-37)沉淀1.3kg，收率80％。
收率的计算方法是：
收率＝(目标肽质量/目标肽分子量)/(融合蛋白质量/融合蛋白分子量)*100％
实施例18、融合蛋白PTH1-N(SEQ ID NO.26)的酶切
将实施例4所得的15.6kg融合蛋白溶液经0.22μm的滤膜过滤后加入灭菌后的30L的反应釜中，维持温度22℃左右，加入肠激酶液45ml(3000μ/ml)，缓慢搅拌反应，HPLC监控反应进度，8h后反应结束。用2N盐酸调体系pH至6.8，得目的多肽溶液，收率85％。
实施例19、其它一些融合蛋白的酶切
重组融合蛋白SEQ ID NO.5-SEQ ID NO.23的酶切参照实施例17进行，重组融合蛋白SEQ ID NO.24、SEQ ID NO.25、SEQ ID NO.27-SEQ ID NO.33的酶切参照实施例18的条件进行。
SEQ ID NO.4～7，10，11，16，17,22～27，30，33所示融合蛋白酶切的HPLC图谱如图3～17所示。
实施例20、目标多肽Arg34-GLP-1(7-37)的活性检测
1.方法
GLP-1是已发现的促胰岛素分泌作用最强的肠肽类激素，GLP1R是GLP-1的受体，其与激动剂结合发挥作用。激动剂结合GLP1R后，激活细胞膜内环腺苷酸(cAMP)。
将实施例17制备得到的目标多肽Arg34-GLP-1(7-37)、重组融合蛋白mTrA-(R34)7-37(SEQ ID NO.4)以及利拉鲁肽对照品作为待检样品，通过AlphaScreen_cAMP试剂盒检测细胞内cAMP的水平来判定化合物的活性。
2.结果
结果如表1所示。
本发明制备得到的目标多肽具备良好的生物活性。
表1活性检测结果
注：Top，量效曲线的顶；Bottom，量效曲线的底；Slope，量效曲线的斜率；RelativeEC50，半数效应浓度。
本发明的制备多肽的方法十分便利，设备要求低，破胞快速，成本低，所得蛋白收率高，活性高，非常适合大规模生产。
SEQUENCE LISTING
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Lys Val Lys Ile Val Lys Val Asn Val Asp Glu Asn Gln Glu Thr Ala
50 55 60
Ser Lys Phe Gly Val Met Ser Ile Pro Thr Leu Leu Val Phe Lys Asn
65 70 75 80
Gly Glu Leu Val Asp Lys Ala Val Gly Tyr Gln Pro Lys Glu Ala Leu
85 90 95
Val Gln Leu Val Gly Lys His Val Gly Ser Gly Ser Gly His His His
100 105 110
His His His Asp Asp Asp Asp Lys His Ala Glu Gly Thr Phe Thr Ser
115 120 125
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
130 135 140
Trp Leu Val Arg Gly Arg Gly
145 150
<210> 9
<211> 146
<212> PRT
<213> 人工序列(artificial sequence)
<400> 9
Met Ala Ile Val Asn Ala Thr Asp Gln Thr Phe Ala Ala Glu Thr Lys
1 5 10 15
Asp Gly Leu Thr Leu Val Asp Phe Trp Ala Pro Trp Ser Gly Pro Cys
20 25 30
Arg Met Ile Ala Pro Val Leu Glu Glu Leu Asp Arg Glu Met Gly Asp
35 40 45
Lys Val Lys Ile Val Lys Val Asn Val Asp Glu Asn Gln Glu Thr Ala
50 55 60
Ser Lys Phe Gly Val Met Ser Ile Pro Thr Leu Leu Val Phe Lys Asn
65 70 75 80
Gly Glu Leu Val Asp Lys Ala Val Gly Tyr Gln Pro Lys Glu Ala Leu
85 90 95
Val Gln Leu Val Gly Lys His Val His His His His His His Asp Asp
100 105 110
Asp Asp Lys His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
115 120 125
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
130 135 140
Arg Gly
145
<210> 10
<211> 141
<212> PRT
<213> 人工序列(artificial sequence)
<400> 10
Met Ala Ile Val Asn Ala Thr Asp Gln Thr Phe Ala Ala Glu Thr Lys
1 5 10 15
Asp Gly Leu Thr Leu Val Asp Phe Trp Ala Pro Trp Ser Gly Pro Cys
20 25 30
Arg Met Ile Ala Pro Val Leu Glu Glu Leu Asp Arg Glu Met Gly Asp
35 40 45
Lys Val Lys Ile Val Lys Val Asn Val Asp Glu Asn Gln Glu Thr Ala
50 55 60
Ser Lys Phe Gly Val Met Ser Ile Pro Thr Leu Leu Val Phe Lys Asn
65 70 75 80
Gly Glu Leu Val Asp Lys Ala Val Gly Tyr Gln Pro Lys Glu Ala Leu
85 90 95
Val Gln Leu Val Gly Lys His Val Gly Ser Gly Asp Asp Asp Asp Lys
100 105 110
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala
115 120 125
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
130 135 140
<210> 11
<211> 143
<212> PRT
<213> 人工序列(artificial sequence)
<400> 11
Met Ala Ile Val Asn Ala Thr Asp Gln Thr Phe Ala Ala Glu Thr Lys
1 5 10 15
Asp Gly Leu Thr Leu Val Asp Phe Trp Ala Pro Trp Ser Gly Pro Cys
20 25 30
Arg Met Ile Ala Pro Val Leu Glu Glu Leu Asp Arg Glu Met Gly Asp
35 40 45
Lys Val Lys Ile Val Lys Val Asn Val Asp Glu Asn Gln Glu Thr Ala
50 55 60
Ser Lys Phe Gly Val Met Ser Ile Pro Thr Leu Leu Val Phe Lys Asn
65 70 75 80
Gly Glu Leu Val Asp Lys Ala Val Gly Tyr Gln Pro Lys Glu Ala Leu
85 90 95
Val Gln Leu Val Gly Lys His Val Gly Ser Gly Ser Gly Asp Asp Asp
100 105 110
Asp Lys Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
115 120 125
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
130 135 140
<210> 12
<211> 138
<212> PRT
<213> 人工序列(artificial sequence)
<400> 12
Met Ala Ile Val Asn Ala Thr Asp Gln Thr Phe Ala Ala Glu Thr Lys
1 5 10 15
Asp Gly Leu Thr Leu Val Asp Phe Trp Ala Pro Trp Ser Gly Pro Cys
20 25 30
Arg Met Ile Ala Pro Val Leu Glu Glu Leu Asp Arg Glu Met Gly Asp
35 40 45
Lys Val Lys Ile Val Lys Val Asn Val Asp Glu Asn Gln Glu Thr Ala
50 55 60
Ser Lys Phe Gly Val Met Ser Ile Pro Thr Leu Leu Val Phe Lys Asn
65 70 75 80
Gly Glu Leu Val Asp Lys Ala Val Gly Tyr Gln Pro Lys Glu Ala Leu
85 90 95
Val Gln Leu Val Gly Lys His Val Asp Asp Asp Asp Lys Glu Gly Thr
100 105 110
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu
115 120 125
Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
130 135
<210> 13
<211> 147
<212> PRT
<213> 人工序列(artificial sequence)
<400> 13
Met Ala Ile Val Asn Ala Thr Asp Gln Thr Phe Ala Ala Glu Thr Lys
1 5 10 15
Asp Gly Leu Thr Leu Val Asp Phe Trp Ala Pro Trp Ser Gly Pro Cys
20 25 30
Arg Met Ile Ala Pro Val Leu Glu Glu Leu Asp Arg Glu Met Gly Asp
35 40 45
Lys Val Lys Ile Val Lys Val Asn Val Asp Glu Asn Gln Glu Thr Ala
50 55 60
Ser Lys Phe Gly Val Met Ser Ile Pro Thr Leu Leu Val Phe Lys Asn
65 70 75 80
Gly Glu Leu Val Asp Lys Ala Val Gly Tyr Gln Pro Lys Glu Ala Leu
85 90 95
Val Gln Leu Val Gly Lys His Val Gly Ser Gly His His His His His
100 105 110
His Asp Asp Asp Asp Lys Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
115 120 125
Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg
130 135 140
Gly Arg Gly
145
<210> 14
<211> 149
<212> PRT
<213> 人工序列(artificial sequence)
<400> 14
Met Ala Ile Val Asn Ala Thr Asp Gln Thr Phe Ala Ala Glu Thr Lys
1 5 10 15
Asp Gly Leu Thr Leu Val Asp Phe Trp Ala Pro Trp Ser Gly Pro Cys
20 25 30
Arg Met Ile Ala Pro Val Leu Glu Glu Leu Asp Arg Glu Met Gly Asp
35 40 45
Lys Val Lys Ile Val Lys Val Asn Val Asp Glu Asn Gln Glu Thr Ala
50 55 60
Ser Lys Phe Gly Val Met Ser Ile Pro Thr Leu Leu Val Phe Lys Asn
65 70 75 80
Gly Glu Leu Val Asp Lys Ala Val Gly Tyr Gln Pro Lys Glu Ala Leu
85 90 95
Val Gln Leu Val Gly Lys His Val Gly Ser Gly Ser Gly His His His
100 105 110
His His His Asp Asp Asp Asp Lys Glu Gly Thr Phe Thr Ser Asp Val
115 120 125
Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
130 135 140
Val Arg Gly Arg Gly
145
<210> 15
<211> 144
<212> PRT
<213> 人工序列(artificial sequence)
<400> 15
Met Ala Ile Val Asn Ala Thr Asp Gln Thr Phe Ala Ala Glu Thr Lys
1 5 10 15
Asp Gly Leu Thr Leu Val Asp Phe Trp Ala Pro Trp Ser Gly Pro Cys
20 25 30
Arg Met Ile Ala Pro Val Leu Glu Glu Leu Asp Arg Glu Met Gly Asp
35 40 45
Lys Val Lys Ile Val Lys Val Asn Val Asp Glu Asn Gln Glu Thr Ala
50 55 60
Ser Lys Phe Gly Val Met Ser Ile Pro Thr Leu Leu Val Phe Lys Asn
65 70 75 80
Gly Glu Leu Val Asp Lys Ala Val Gly Tyr Gln Pro Lys Glu Ala Leu
85 90 95
Val Gln Leu Val Gly Lys His Val His His His His His His Asp Asp
100 105 110
Asp Asp Lys Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
115 120 125
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
130 135 140
<210> 16
<211> 139
<212> PRT
<213> 人工序列(artificial sequence)
<400> 16
Met Ala Ile Val Asn Ala Thr Asp Gln Thr Phe Ala Ala Glu Thr Lys
1 5 10 15
Asp Gly Leu Thr Leu Val Asp Phe Trp Ala Pro Trp Ser Gly Pro Cys
20 25 30
Arg Met Ile Ala Pro Val Leu Glu Glu Leu Asp Arg Glu Met Gly Asp
35 40 45
Lys Val Lys Ile Val Lys Val Asn Val Asp Glu Asn Gln Glu Thr Ala
50 55 60
Ser Lys Phe Gly Val Met Ser Ile Pro Thr Leu Leu Val Phe Lys Asn
65 70 75 80
Gly Glu Leu Val Asp Lys Ala Val Gly Tyr Gln Pro Lys Glu Ala Leu
85 90 95
Val Gln Leu Val Gly Lys His Val Gly Ser Gly Asp Asp Asp Asp Lys
100 105 110
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys
115 120 125
Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
130 135
<210> 17
<211> 141
<212> PRT
<213> 人工序列(artificial sequence)
<400> 17
Met Ala Ile Val Asn Ala Thr Asp Gln Thr Phe Ala Ala Glu Thr Lys
1 5 10 15
Asp Gly Leu Thr Leu Val Asp Phe Trp Ala Pro Trp Ser Gly Pro Cys
20 25 30
Arg Met Ile Ala Pro Val Leu Glu Glu Leu Asp Arg Glu Met Gly Asp
35 40 45
Lys Val Lys Ile Val Lys Val Asn Val Asp Glu Asn Gln Glu Thr Ala
50 55 60
Ser Lys Phe Gly Val Met Ser Ile Pro Thr Leu Leu Val Phe Lys Asn
65 70 75 80
Gly Glu Leu Val Asp Lys Ala Val Gly Tyr Gln Pro Lys Glu Ala Leu
85 90 95
Val Gln Leu Val Gly Lys His Val Gly Ser Gly Ser Gly Asp Asp Asp
100 105 110
Asp Lys Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala
115 120 125
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
130 135 140
<210> 18
<211> 136
<212> PRT
<213> 人工序列(artificial sequence)
<400> 18
Met Ala Ile Val Asn Ala Thr Asp Gln Thr Phe Ala Ala Glu Thr Lys
1 5 10 15
Asp Gly Leu Thr Leu Val Asp Phe Trp Ala Pro Trp Ser Gly Pro Cys
20 25 30
Arg Met Ile Ala Pro Val Leu Glu Glu Leu Asp Arg Glu Met Gly Asp
35 40 45
Lys Val Lys Ile Val Lys Val Asn Val Asp Glu Asn Gln Glu Thr Ala
50 55 60
Ser Lys Phe Gly Val Met Ser Ile Pro Thr Leu Leu Val Phe Lys Asn
65 70 75 80
Gly Glu Leu Val Asp Lys Ala Val Gly Tyr Gln Pro Lys Glu Ala Leu
85 90 95
Val Gln Leu Val Gly Lys His Val Asp Asp Asp Asp Lys Thr Phe Thr
100 105 110
Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile
115 120 125
Ala Trp Leu Val Arg Gly Arg Gly
130 135
<210> 19
<211> 145
<212> PRT
<213> 人工序列(artificial sequence)
<400> 19
Met Ala Ile Val Asn Ala Thr Asp Gln Thr Phe Ala Ala Glu Thr Lys
1 5 10 15
Asp Gly Leu Thr Leu Val Asp Phe Trp Ala Pro Trp Ser Gly Pro Cys
20 25 30
Arg Met Ile Ala Pro Val Leu Glu Glu Leu Asp Arg Glu Met Gly Asp
35 40 45
Lys Val Lys Ile Val Lys Val Asn Val Asp Glu Asn Gln Glu Thr Ala
50 55 60
Ser Lys Phe Gly Val Met Ser Ile Pro Thr Leu Leu Val Phe Lys Asn
65 70 75 80
Gly Glu Leu Val Asp Lys Ala Val Gly Tyr Gln Pro Lys Glu Ala Leu
85 90 95
Val Gln Leu Val Gly Lys His Val Gly Ser Gly His His His His His
100 105 110
His Asp Asp Asp Asp Lys Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
115 120 125
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
130 135 140
Gly
145
<210> 20
<211> 147
<212> PRT
<213> 人工序列(artificial sequence)
<400> 20
Met Ala Ile Val Asn Ala Thr Asp Gln Thr Phe Ala Ala Glu Thr Lys
1 5 10 15
Asp Gly Leu Thr Leu Val Asp Phe Trp Ala Pro Trp Ser Gly Pro Cys
20 25 30
Arg Met Ile Ala Pro Val Leu Glu Glu Leu Asp Arg Glu Met Gly Asp
35 40 45
Lys Val Lys Ile Val Lys Val Asn Val Asp Glu Asn Gln Glu Thr Ala
50 55 60
Ser Lys Phe Gly Val Met Ser Ile Pro Thr Leu Leu Val Phe Lys Asn
65 70 75 80
Gly Glu Leu Val Asp Lys Ala Val Gly Tyr Gln Pro Lys Glu Ala Leu
85 90 95
Val Gln Leu Val Gly Lys His Val Gly Ser Gly Ser Gly His His His
100 105 110
His His His Asp Asp Asp Asp Lys Thr Phe Thr Ser Asp Val Ser Ser
115 120 125
Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg
130 135 140
Gly Arg Gly
145
<210> 21
<211> 142
<212> PRT
<213> 人工序列(artificial sequence)
<400> 21
Met Ala Ile Val Asn Ala Thr Asp Gln Thr Phe Ala Ala Glu Thr Lys
1 5 10 15
Asp Gly Leu Thr Leu Val Asp Phe Trp Ala Pro Trp Ser Gly Pro Cys
20 25 30
Arg Met Ile Ala Pro Val Leu Glu Glu Leu Asp Arg Glu Met Gly Asp
35 40 45
Lys Val Lys Ile Val Lys Val Asn Val Asp Glu Asn Gln Glu Thr Ala
50 55 60
Ser Lys Phe Gly Val Met Ser Ile Pro Thr Leu Leu Val Phe Lys Asn
65 70 75 80
Gly Glu Leu Val Asp Lys Ala Val Gly Tyr Gln Pro Lys Glu Ala Leu
85 90 95
Val Gln Leu Val Gly Lys His Val His His His His His His Asp Asp
100 105 110
Asp Asp Lys Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
115 120 125
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
130 135 140
<210> 22
<211> 145
<212> PRT
<213> 人工序列(artificial sequence)
<400> 22
Met Ala Ile Val Asn Ala Thr Asp Gln Thr Phe Ala Ala Glu Thr Lys
1 5 10 15
Asp Gly Leu Thr Leu Val Asp Phe Trp Ala Pro Trp Cys Gly Pro Cys
20 25 30
Arg Met Ile Ala Pro Val Leu Glu Glu Leu Asp Arg Glu Met Gly Asp
35 40 45
Lys Val Lys Ile Val Lys Val Asn Val Asp Glu Asn Gln Glu Thr Ala
50 55 60
Ser Lys Phe Gly Val Met Ser Ile Pro Thr Leu Leu Val Phe Lys Asn
65 70 75 80
Gly Glu Leu Val Asp Lys Ala Val Gly Tyr Gln Pro Lys Glu Ala Leu
85 90 95
Val Gln Leu Val Gly Lys His Val Gly Ser Gly Asp Asp Asp Asp Lys
100 105 110
His Gly Asp Gly Ser Phe Ser Asp Glu Met Asn Thr Ile Leu Asp Asn
115 120 125
Leu Ala Ala Arg Asp Phe Ile Asn Trp Leu Ile Gln Thr Lys Ile Thr
130 135 140
Asp
145
<210> 23
<211> 151
<212> PRT
<213> 人工序列(artificial sequence)
<400> 23
Met Ala Ile Val Asn Ala Thr Asp Gln Thr Phe Ala Ala Glu Thr Lys
1 5 10 15
Asp Gly Leu Thr Leu Val Asp Phe Trp Ala Pro Trp Cys Gly Pro Cys
20 25 30
Arg Met Ile Ala Pro Val Leu Glu Glu Leu Asp Arg Glu Met Gly Asp
35 40 45
Lys Val Lys Ile Val Lys Val Asn Val Asp Glu Asn Gln Glu Thr Ala
50 55 60
Ser Lys Phe Gly Val Met Ser Ile Pro Thr Leu Leu Val Phe Lys Asn
65 70 75 80
Gly Glu Leu Val Asp Lys Ala Val Gly Tyr Gln Pro Lys Glu Ala Leu
85 90 95
Val Gln Leu Val Gly Lys His Val Gly Ser Gly His His His His His
100 105 110
His Asp Asp Asp Asp Lys His Gly Asp Gly Ser Phe Ser Asp Glu Met
115 120 125
Asn Thr Ile Leu Asp Asn Leu Ala Ala Arg Asp Phe Ile Asn Trp Leu
130 135 140
Ile Gln Thr Lys Ile Thr Asp
145 150
<210> 24
<211> 152
<212> PRT
<213> 人工序列(artificial sequence)
<400> 24
Met Ala Ile Val Asn Ala Thr Asp Gln Thr Phe Ala Ala Glu Thr Lys
1 5 10 15
Asp Gly Leu Thr Leu Val Asp Phe Trp Ala Pro Trp Cys Gly Pro Cys
20 25 30
Arg Met Ile Ala Pro Val Leu Glu Glu Leu Asp Arg Glu Met Gly Asp
35 40 45
Lys Val Lys Ile Val Lys Val Asn Val Asp Glu Asn Gln Glu Thr Ala
50 55 60
Ser Lys Phe Gly Val Met Ser Ile Pro Thr Leu Leu Val Phe Lys Asn
65 70 75 80
Gly Glu Leu Val Asp Lys Ala Val Gly Tyr Gln Pro Lys Glu Ala Leu
85 90 95
Val Gln Leu Val Gly Lys His Val Gly Ser Gly His His His His His
100 105 110
His Asp Asp Asp Asp Lys Ser Val Ser Glu Ile Gln Leu Met His Asn
115 120 125
Leu Gly Lys His Leu Asn Ser Met Glu Arg Val Glu Trp Leu Arg Lys
130 135 140
Lys Leu Gln Asp Val His Asn Phe
145 150
<210> 25
<211> 146
<212> PRT
<213> 人工序列(artificial sequence)
<400> 25
Met Ala Ile Val Asn Ala Thr Asp Gln Thr Phe Ala Ala Glu Thr Lys
1 5 10 15
Asp Gly Leu Thr Leu Val Asp Phe Trp Ala Pro Trp Cys Gly Pro Cys
20 25 30
Arg Met Ile Ala Pro Val Leu Glu Glu Leu Asp Arg Glu Met Gly Asp
35 40 45
Lys Val Lys Ile Val Lys Val Asn Val Asp Glu Asn Gln Glu Thr Ala
50 55 60
Ser Lys Phe Gly Val Met Ser Ile Pro Thr Leu Leu Val Phe Lys Asn
65 70 75 80
Gly Glu Leu Val Asp Lys Ala Val Gly Tyr Gln Pro Lys Glu Ala Leu
85 90 95
Val Gln Leu Val Gly Lys His Val Gly Ser Gly Asp Asp Asp Asp Lys
100 105 110
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
115 120 125
Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His
130 135 140
Asn Phe
145
<210> 26
<211> 171
<212> PRT
<213> 人工序列(artificial sequence)
<400> 26
Met Tyr Gly Asp Glu Leu Asp Phe Tyr Thr Lys Met Tyr Asn Glu Ile
1 5 10 15
Glu Arg Leu Arg Lys Glu Gly Val Val Glu Tyr Val Thr Ser Tyr Asp
20 25 30
Arg Phe Arg Glu Ile Ile Glu Asn His Pro Val Val Val Ala Val Phe
35 40 45
Thr Thr Pro Thr Cys Ser Ala Cys Met Ile Tyr Lys Pro Ile Phe Tyr
50 55 60
Met Val Ala Glu Lys Leu Arg Asp Lys Ala Lys Trp Ile Glu Val Asp
65 70 75 80
Ala Tyr Glu Ala Pro Glu Ala Ala Phe Glu Ser Gly Val Thr Ala Thr
85 90 95
Pro Thr Thr Ile Val Tyr Val Asp Gly Glu Ala Val Asp Gly Phe Val
100 105 110
Gly Ile Leu Asp Glu Glu Gly Leu Val Glu Ile Val Lys Gln Tyr Val
115 120 125
Glu Glu Gln Ser Asp Asp Asp Asp Lys Ser Val Ser Glu Ile Gln Leu
130 135 140
Met His Asn Leu Gly Lys His Leu Asn Ser Met Glu Arg Val Glu Trp
145 150 155 160
Leu Arg Lys Lys Leu Gln Asp Val His Asn Phe
165 170
<210> 27
<211> 140
<212> PRT
<213> 人工序列(artificial sequence)
<400> 27
Met Ala Ile Val Asn Ala Thr Asp Gln Thr Phe Ala Ala Glu Thr Lys
1 5 10 15
Asp Gly Leu Thr Leu Val Asp Phe Trp Ala Pro Trp Ser Gly Pro Cys
20 25 30
Arg Met Ile Ala Pro Val Leu Glu Glu Leu Asp Arg Glu Met Gly Asp
35 40 45
Lys Val Lys Ile Val Lys Val Asn Val Asp Glu Asn Gln Glu Thr Ala
50 55 60
Ser Lys Phe Gly Val Met Ser Ile Pro Thr Leu Leu Val Phe Lys Asn
65 70 75 80
Gly Glu Leu Val Asp Lys Ala Val Gly Tyr Gln Pro Lys Glu Ala Leu
85 90 95
Val Gln Leu Val Gly Lys His Val Gly Ser Gly Asp Asp Asp Asp Lys
100 105 110
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln
115 120 125
Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu
130 135 140
<210> 28
<211> 142
<212> PRT
<213> 人工序列(artificial sequence)
<400> 28
Met Ala Ile Val Asn Ala Thr Asp Gln Thr Phe Ala Ala Glu Thr Lys
1 5 10 15
Asp Gly Leu Thr Leu Val Asp Phe Trp Ala Pro Trp Ser Gly Pro Cys
20 25 30
Arg Met Ile Ala Pro Val Leu Glu Glu Leu Asp Arg Glu Met Gly Asp
35 40 45
Lys Val Lys Ile Val Lys Val Asn Val Asp Glu Asn Gln Glu Thr Ala
50 55 60
Ser Lys Phe Gly Val Met Ser Ile Pro Thr Leu Leu Val Phe Lys Asn
65 70 75 80
Gly Glu Leu Val Asp Lys Ala Val Gly Tyr Gln Pro Lys Glu Ala Leu
85 90 95
Val Gln Leu Val Gly Lys His Val Gly Ser Gly Ser Gly Asp Asp Asp
100 105 110
Asp Lys Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser
115 120 125
Ile Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu
130 135 140
<210> 29
<211> 137
<212> PRT
<213> 人工序列(artificial sequence)
<400> 29
Met Ala Ile Val Asn Ala Thr Asp Gln Thr Phe Ala Ala Glu Thr Lys
1 5 10 15
Asp Gly Leu Thr Leu Val Asp Phe Trp Ala Pro Trp Ser Gly Pro Cys
20 25 30
Arg Met Ile Ala Pro Val Leu Glu Glu Leu Asp Arg Glu Met Gly Asp
35 40 45
Lys Val Lys Ile Val Lys Val Asn Val Asp Glu Asn Gln Glu Thr Ala
50 55 60
Ser Lys Phe Gly Val Met Ser Ile Pro Thr Leu Leu Val Phe Lys Asn
65 70 75 80
Gly Glu Leu Val Asp Lys Ala Val Gly Tyr Gln Pro Lys Glu Ala Leu
85 90 95
Val Gln Leu Val Gly Lys His Val Asp Asp Asp Asp Lys Ala Val Ser
100 105 110
Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln Asp Leu Arg
115 120 125
Arg Arg Glu Leu Leu Glu Lys Leu Leu
130 135
<210> 30
<211> 151
<212> PRT
<213> 人工序列(artificial sequence)
<400> 30
Met Ala Ile Val Asn Ala Thr Asp Gln Thr Phe Ala Ala Glu Thr Lys
1 5 10 15
Asp Gly Leu Thr Leu Val Asp Phe Trp Ala Pro Trp Ser Gly Pro Cys
20 25 30
Arg Met Ile Ala Pro Val Leu Glu Glu Leu Asp Arg Glu Met Gly Asp
35 40 45
Lys Val Lys Ile Val Lys Val Asn Val Asp Glu Asn Gln Glu Thr Ala
50 55 60
Ser Lys Phe Gly Val Met Ser Ile Pro Thr Leu Leu Val Phe Lys Asn
65 70 75 80
Gly Glu Leu Val Asp Lys Ala Val Gly Tyr Gln Pro Lys Glu Ala Leu
85 90 95
Val Gln Leu Val Gly Lys His Val Gly Ser Gly Asp Asp Asp Asp Lys
100 105 110
His Gly Glu Gly Thr Phe Ser Ser Glu Leu Ala Thr Ile Leu Asp Ala
115 120 125
Leu Ala Ala Arg Asp Phe Ile Ala Trp Leu Ile Ala Thr Lys Ile Thr
130 135 140
Asp Lys Lys Lys Lys Lys Lys
145 150
<210> 31
<211> 153
<212> PRT
<213> 人工序列(artificial sequence)
<400> 31
Met Ala Ile Val Asn Ala Thr Asp Gln Thr Phe Ala Ala Glu Thr Lys
1 5 10 15
Asp Gly Leu Thr Leu Val Asp Phe Trp Ala Pro Trp Ser Gly Pro Cys
20 25 30
Arg Met Ile Ala Pro Val Leu Glu Glu Leu Asp Arg Glu Met Gly Asp
35 40 45
Lys Val Lys Ile Val Lys Val Asn Val Asp Glu Asn Gln Glu Thr Ala
50 55 60
Ser Lys Phe Gly Val Met Ser Ile Pro Thr Leu Leu Val Phe Lys Asn
65 70 75 80
Gly Glu Leu Val Asp Lys Ala Val Gly Tyr Gln Pro Lys Glu Ala Leu
85 90 95
Val Gln Leu Val Gly Lys His Val Gly Ser Gly Ser Gly Asp Asp Asp
100 105 110
Asp Lys His Gly Glu Gly Thr Phe Ser Ser Glu Leu Ala Thr Ile Leu
115 120 125
Asp Ala Leu Ala Ala Arg Asp Phe Ile Ala Trp Leu Ile Ala Thr Lys
130 135 140
Ile Thr Asp Lys Lys Lys Lys Lys Lys
145 150
<210> 32
<211> 148
<212> PRT
<213> 人工序列(artificial sequence)
<400> 32
Met Ala Ile Val Asn Ala Thr Asp Gln Thr Phe Ala Ala Glu Thr Lys
1 5 10 15
Asp Gly Leu Thr Leu Val Asp Phe Trp Ala Pro Trp Ser Gly Pro Cys
20 25 30
Arg Met Ile Ala Pro Val Leu Glu Glu Leu Asp Arg Glu Met Gly Asp
35 40 45
Lys Val Lys Ile Val Lys Val Asn Val Asp Glu Asn Gln Glu Thr Ala
50 55 60
Ser Lys Phe Gly Val Met Ser Ile Pro Thr Leu Leu Val Phe Lys Asn
65 70 75 80
Gly Glu Leu Val Asp Lys Ala Val Gly Tyr Gln Pro Lys Glu Ala Leu
85 90 95
Val Gln Leu Val Gly Lys His Val Asp Asp Asp Asp Lys His Gly Glu
100 105 110
Gly Thr Phe Ser Ser Glu Leu Ala Thr Ile Leu Asp Ala Leu Ala Ala
115 120 125
Arg Asp Phe Ile Ala Trp Leu Ile Ala Thr Lys Ile Thr Asp Lys Lys
130 135 140
Lys Lys Lys Lys
145
<210> 33
<211> 128
<212> PRT
<213> 人工序列(artificial sequence)
<400> 33
Met Ala Ile Val Asn Ala Thr Asp Gln Thr Phe Ala Ala Glu Thr Lys
1 5 10 15
Asp Gly Leu Thr Leu Val Asp Phe Trp Ala Pro Trp Ser Gly Pro Cys
20 25 30
Arg Met Ile Ala Pro Val Leu Glu Glu Leu Asp Arg Glu Met Gly Asp
35 40 45
Lys Val Lys Ile Val Lys Val Asn Val Asp Glu Asn Gln Glu Thr Ala
50 55 60
Ser Lys Phe Gly Val Met Ser Ile Pro Thr Leu Leu Val Phe Lys Asn
65 70 75 80
Gly Glu Leu Val Asp Lys Ala Val Gly Tyr Gln Pro Lys Glu Ala Leu
85 90 95
Val Gln Leu Val Gly Lys His Val Gly Ser Gly Asp Asp Asp Asp Lys
100 105 110
Asn Asp Glu Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu
115 120 125
<210> 34
<211> 130
<212> PRT
<213> 人工序列(artificial sequence)
<400> 34
Met Ala Ile Val Asn Ala Thr Asp Gln Thr Phe Ala Ala Glu Thr Lys
1 5 10 15
Asp Gly Leu Thr Leu Val Asp Phe Trp Ala Pro Trp Ser Gly Pro Cys
20 25 30
Arg Met Ile Ala Pro Val Leu Glu Glu Leu Asp Arg Glu Met Gly Asp
35 40 45
Lys Val Lys Ile Val Lys Val Asn Val Asp Glu Asn Gln Glu Thr Ala
50 55 60
Ser Lys Phe Gly Val Met Ser Ile Pro Thr Leu Leu Val Phe Lys Asn
65 70 75 80
Gly Glu Leu Val Asp Lys Ala Val Gly Tyr Gln Pro Lys Glu Ala Leu
85 90 95
Val Gln Leu Val Gly Lys His Val Gly Ser Gly Ser Gly Asp Asp Asp
100 105 110
Asp Lys Asn Asp Glu Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly
115 120 125
Cys Leu
130
<210> 35
<211> 125
<212> PRT
<213> 人工序列(artificial sequence)
<400> 35
Met Ala Ile Val Asn Ala Thr Asp Gln Thr Phe Ala Ala Glu Thr Lys
1 5 10 15
Asp Gly Leu Thr Leu Val Asp Phe Trp Ala Pro Trp Ser Gly Pro Cys
20 25 30
Arg Met Ile Ala Pro Val Leu Glu Glu Leu Asp Arg Glu Met Gly Asp
35 40 45
Lys Val Lys Ile Val Lys Val Asn Val Asp Glu Asn Gln Glu Thr Ala
50 55 60
Ser Lys Phe Gly Val Met Ser Ile Pro Thr Leu Leu Val Phe Lys Asn
65 70 75 80
Gly Glu Leu Val Asp Lys Ala Val Gly Tyr Gln Pro Lys Glu Ala Leu
85 90 95
Val Gln Leu Val Gly Lys His Val Asp Asp Asp Asp Lys Asn Asp Glu
100 105 110
Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu
115 120 125
<210> 36
<211> 31
<212> PRT
<213> 人工序列(artificial sequence)
<400> 36
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
20 25 30
<210> 37
<211> 29
<212> PRT
<213> 人工序列(artificial sequence)
<400> 37
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala
1 5 10 15
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
20 25
<210> 38
<211> 27
<212> PRT
<213> 人工序列(artificial sequence)
<400> 38
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys
1 5 10 15
Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
20 25
<210> 39
<211> 33
<212> PRT
<213> 人工序列(artificial sequence)
<400> 39
His Gly Asp Gly Ser Phe Ser Asp Glu Met Asn Thr Ile Leu Asp Asn
1 5 10 15
Leu Ala Ala Arg Asp Phe Ile Asn Trp Leu Ile Gln Thr Lys Ile Thr
20 25 30
Asp
<210> 40
<211> 34
<212> PRT
<213> 人工序列(artificial sequence)
<400> 40
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His
20 25 30
Asn Phe
<210> 41
<211> 28
<212> PRT
<213> 人工序列(artificial sequence)
<400> 41
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln
1 5 10 15
Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu
20 25
<210> 42
<211> 39
<212> PRT
<213> 人工序列(artificial sequence)
<400> 42
His Gly Glu Gly Thr Phe Ser Ser Glu Leu Ala Thr Ile Leu Asp Ala
1 5 10 15
Leu Ala Ala Arg Asp Phe Ile Ala Trp Leu Ile Ala Thr Lys Ile Thr
20 25 30
Asp Lys Lys Lys Lys Lys Lys
35
<210> 43
<211> 16
<212> PRT
<213> 人工序列(artificial sequence)
<400> 43
Asn Asp Glu Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu
1 5 10 15

Claims (11)

1.一种利用热稳定融合蛋白制备多肽的方法，其特征在于，它包括如下步骤：
1)使用重组大肠杆菌发酵表达融合蛋白，该融合蛋白是热稳定蛋白与目标多肽的融合蛋白；
2)将破胞缓冲液加热到60～100℃，与发酵液按质量比1∶(0.5～4)混合，同时加入相当于混合液质量0.01％-2％的表面活性剂，搅拌破胞；
3)离心取上清；
4)过滤除杂，超滤浓缩；
5)酶切，得目标多肽；
步骤1)所述热稳定蛋白是在60～100℃高温下维持稳定的蛋白，所述融合蛋白也能在60～100℃高温下维持稳定；
步骤2)中，表面活性剂是非离子型表面活性剂；
步骤2)中，所述破胞缓冲液能使破胞后混合溶液的pH值与融合蛋白等电点的差值的绝对值在2以上。
2.如权利要求1所述的制备多肽的方法，其特征在于：
步骤2)中的表面活性剂是聚醚类、聚乙二醇类、酯类或多元醇类非离子型表面活性剂，优选地，为曲拉通X-100或吐温-80。
3.如权利要求1所述的制备多肽的方法，其特征在于：
步骤2)中的盐类缓冲液，所述的盐类选自磷酸盐、Tris-HCl、乙酸盐；和/或，浓度范围是10-100mM。
4.如权利要求1所述的制备多肽的方法，其特征在于：
步骤3)所述离心为连续离心，优选地，为碟片式离心。
5.如权利要求1所述的制备多肽的方法，其特征在于：
步骤3)中离心后的上清液中不溶物湿重不超过上清液的3％，优选地，不超过上清液的1％。
6.如权利要求1所述的制备多肽的方法，其特征在于：
步骤4)中，过滤除杂使用PP材质的中空纤维膜或PES材质的微滤膜；和/或，超滤浓缩采用的膜是PES材质的。
7.如权利要求1所述的制备多肽的方法，其特征在于：
步骤4)的超滤过程中，料液温度不超过40℃；和/或，压力不超过0.4MPa；和/或，pH值不超过8.5；
优选地，步骤4)的超滤过程中，料液温度不超过35℃；和/或，压力补偿不超过过0.2MPa；和/或，pH值不超过7.8。
8.如权利要求1所述的制备多肽的方法，其特征在于：
步骤5)中的酶是肠激酶、胰蛋白酶或赖氨酰肽链内切酶，优选地，为肠激酶。
9.如权利要求1所述的制备多肽的方法，其特征在于：
步骤5)中反应在水相或者较低比例有机相体系中进行；
所述有机相是乙腈或者短链醇类；所述短链指的是分子中碳原子个数不超过3；
所述较低比例指比例不超过25％，优选地不超过10％，最优选地是低于5％。
10.如权利要求1所述的制备多肽的方法，其特征在于：
步骤1)所述热稳定蛋白序列如SEQ ID NO.1～3中任一所示；
和/或，目标多肽的序列如SEQ ID NO.36～43中任一所示；
和/或，融合蛋白中，热稳定蛋白与目标多肽之间还有一段连接肽；
优选地，融合蛋白的序列如SEQ ID NO.4～25、SEQ ID NO.27～35任一所示。
11.如权利要求1所述的制备多肽的方法，其特征在于：融合蛋白的序列如SEQ IDNO.26所示。
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