CN114381471A - 一种辅助蛋白在重组蛋白生产中的应用及融合表达系统 - Google Patents
一种辅助蛋白在重组蛋白生产中的应用及融合表达系统 Download PDFInfo
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Abstract
本发明提出了一种辅助蛋白在重组蛋白生产中的应用及融合表达系统,所述辅助蛋白的氨基酸序列为SEQ ID NO.1、SEQ ID NO.2、SEQ ID No.3或SEQ ID NO.4,所述的辅助蛋白及包含该辅助蛋白的重组蛋白融合表达系统,表达效率高,能够在细胞内可溶表达重组蛋白,不会形成包涵体,可以通过加热处理菌体实现细胞破碎,释放目标蛋白质,无需超声破碎或高压破碎,操作简单效率高且节约仪器维修和维护成本。加热处理后宿主蛋白会变性形成沉淀而除去,细胞破碎液中目标融合蛋白的含量高、纯度高,且无需多次纯化,大大简化了下游蛋白质的分离纯化工艺,降低纯化成本。
Description
技术领域
本发明涉及生物技术领域,具体而言,涉及一种辅助蛋白在重组蛋白生产中的应用及融合表达系统。
背景技术
重组蛋白表达技术是获取蛋白,研究蛋白质功能、结构,以及筛选靶向药物的重要工具,在基础科学以及医药学领域发挥了重要的作用。大肠杆菌是重要的蛋白质表达系统之一,通过把目标蛋白质的核酸序列与大肠杆菌的基因转录翻译元件组合构建目标蛋白质的重组表达载体,并导入大肠杆菌中,就可以利用大肠杆菌自身的蛋白质表达系统进行目标蛋白质的重组表达。作为一种优秀的蛋白质重组表达系统,大肠杆菌具有很多优点:1)遗传背景清晰,完善的基因操作方法和工具,多组学研究结果以供参考;2)生长繁殖快,几乎每20分钟便可繁殖一代,发酵周期短;3)培养条件简单,通过无机盐培养基即可实现高密度发酵,菌体浓度超过100g/L,目标蛋白质含量最大可达50%,从而获得廉价的重组蛋白质。目前已有上千种蛋白质经大肠杆菌重组表达成功。
然而,大肠杆菌表达系统也仍然存在一些缺陷,例如重组蛋白质基本以胞内表达为主,往往存在蛋白质不可溶而形成包涵体的问题,后续还需繁杂的蛋白质复性过程才能获得有活性功能的目的蛋白质,这会增加工艺流程和分离纯化成本,为了获得目标蛋白质,还需利用超声破碎仪或高压破碎仪等手段对大肠杆菌进行细胞破碎,再从数量众多且复杂的宿主蛋白中分离纯化目标蛋白质,导致下游蛋白质纯化工艺复杂,分离纯化成本较高,破碎设备维护成本也高。且包涵体的纯化过程有时候还会导致目的蛋白的生物活性降低甚至失活,严重影响其应用。
目前部分研究中采用将目标蛋白与分泌信号肽、促溶标签融合表达,与分子伴侣蛋白共表达、降低培养温度等方法,但目标蛋白与分泌信号肽融合表达目前的分泌效率非常低,大部分蛋白质还是存在于细胞内,依然存在形成包涵体和分离纯化过程繁琐的问题,不适合工业放大生产,与促溶标签融合表达能够减少包涵体形成,但后续依然需要繁琐的破碎细胞工艺和从大量的宿主蛋白中分离目标蛋白质,纯化效率低。与分子伴侣共表达能够提高蛋白溶解度,减少包涵体形成,但是其只对部分蛋白表达有效,适用范围较窄。而调整培养条件如降低培养温度等可一定程度降低包涵体的形成,但同时也会降低大肠杆菌的生长速率,影响高密度发酵,同时还会大幅增加动力成本,需要考虑性价比,因此现有技术公开的方法均无法较好的解决大肠杆菌易形成包涵体、蛋白质分离纯化工艺繁杂、蛋白活性受影响等问题。
发明内容
为了解决上述技术问题,本发明提供一种能够有效应用于大肠杆菌表达系统,且产生可溶性表达蛋白、纯化方法简单的重组蛋白生产的融合表达系统及其表达载体。
本发明提供以下技术方案:
一种辅助蛋白在重组蛋白生产中的应用,所述辅助蛋白的氨基酸序列为SEQ IDNO.1、SEQ ID NO.2、SEQ ID No.3或SEQ ID NO.4中的任意一种。
优选地,所述辅助蛋白的氨基酸序列为SEQ ID NO.1。
在一些实施方案中,所述辅助蛋白的核苷酸序列为SEQ ID NO.5、SEQ ID NO.6或SEQ ID No.7中的任意一种,优选地,所述辅助蛋白的核苷酸序列为SEQ ID NO.5。
进一步的,本发明提供一种用于重组蛋白生产的融合表达系统,其包含上述辅助蛋白。
在一些实施方案中,所述融合表达系统包含辅助蛋白-连接肽-切割位点-目标蛋白。
在一些实施方案中,所述连接肽为GSG、GSS、GSGSG、GSGGSG、GSGGGS、GSGGSGG中的一种,优选地,所述连接肽为GSGSG、GSGGSG或GSGGGS。
在一些实施方案中,所述连接肽的1个或多个重复,优选地,所述连接肽为1-3个重复。
在一些实施方案中,所述切割位点为化学切割位点、物理切割位点或蛋白酶切割位点。
在可选的实施方案中,所述化学切割位点为溴化氰(Met↓)、羟胺(Asn↓Gly)等,物理切割位点为低pH(Asp↓Pro)等,蛋白酶切割位点为Xa因子切割位点、凝血酶切割位点、肠激酶切割位点、烟草蚀纹病毒蛋白酶切割位点、凝乳酶切割位点、胶原酶切割位点、3C蛋白酶切割位点、胰蛋白酶切割位点。
上述切割位点优选为蛋白酶切割位点,进一步优选为肠激酶切割位点。
所述切割位点为化学切割位点时,可采用溴化氰(Met↓)、羟胺(Asn↓Gly)等进行切割,所述切割位点为物理切割位点时,可采用低pH(Asp↓Pro)切割,所述切割位点为蛋白酶切割位点时,可采用Xa因子、凝血酶、肠激酶、烟草蚀纹病毒蛋白酶、凝乳酶、胶原酶、3C蛋白酶、胰蛋白酶等切割。
在一些实施方案中,所述目标蛋白为10-200个氨基酸序列,优选地所述目标蛋白为15-100个氨基酸序列,更优选地,所述目标蛋白为胰高血糖素样肽-1、降血压肽、血管紧张肽I和II、抗菌肽等及其衍生多肽、促生长激素神经肽,进一步优选地,所述目标蛋白为胰高血糖素样肽-1。
在优选地实施方案中,本发明所述融合表达系统的氨基酸序列为SEQ ID NO.8中所述的蛋白。
在优选地实施方案中,本发明所述融合表达系统的基因编码序列为SEQ ID NO.9中所述的核苷酸序列。
进一步地,所述融合表达系统中还可以包含纯化标签,所述纯化标签位于辅助蛋白上游或连接肽和切割位点之间,优选地,所述纯化标签为亲和纯化标签。
在一些实施方案中,所述亲和纯化标签为组氨酸标签、赖氨酸标签、精氨酸标签、谷胱甘肽-S-转移酶标签、FLAG标签,进一步优选为组氨酸标签。
在一些优选地实施方案中,本发明所述的融合表达系统的氨基酸序列为SEQ IDNO.10中所述的氨基酸序列。
在优选地实施方案中,本发明所述融合表达系统的基因编码序列为SEQ ID NO.11中所述的核苷酸序列。
在一些实施方案中,本发明所述的表达载体为pET系列、pBAD系列、pTrc系列、pCold系列、pUC系列载体中的一种。在一些实施方案中,本发明所述的载体可以对部分元件进行替换优化,如对启动子进行优化,优选地,所述启动子可以选用T7、lac、Trp、Tac、Trc、λPL、PL、araBAD、GAPDH中的一种或者多种。
进一步的,本发明还提供上述重组表达系统及载体在生产重组融合蛋白中的应用。
进一步的,本发明还提供一种所述重组融合蛋白的制备方法,其步骤为:
1)合成上述的融合蛋白表达基因;
2)将融合蛋白基因插入载体的多克隆位点,构建融合蛋白表达载体;
3)将融合蛋白表达载体导入表达菌株中,构建融合蛋白重组表达工程菌;
4)对融合蛋白重组表达工程菌进行发酵,表达重组融合蛋白;
5)加热处理发酵液,使大肠杆菌裂解释放重组融合蛋白,过滤去除沉淀,收集破碎上清液,得到重组融合蛋白。
优选地,所述加热温度为60-100℃,进一步优选地,所述加热温度为100℃。
优选地,所述加热时间为30-60分钟,进一步优选地,所述加热时间为30分钟。
本发明的有益效果:本发明中所述的辅助蛋白及包含该辅助蛋白的重组蛋白融合表达系统,表达效率高,能够在细胞内可溶表达重组蛋白,不会形成包涵体,可以通过加热处理菌体实现细胞破碎,释放目标蛋白质,无需超声破碎或高压破碎,操作简单效率高且节约仪器维修和维护成本。加热处理后宿主蛋白会变性形成沉淀而除去,细胞破碎液中目标融合蛋白的含量高、纯度高,且无需多次纯化,大大简化了下游蛋白质的分离纯化工艺,降低纯化成本。且对小分子多肽(含15-100个氨基酸)效果特别显著。
附图说明
图1实施例4中SDS-PAGE蛋白电泳图,其中:M为蛋白质标准分子量标记,泳道2为本发明重组融合蛋白工程菌超声破碎离心上清液,泳道3为泳道2样品100℃保温30分钟后离心上清液,泳道4为阴性对照融合蛋白工程菌超声破碎离心上清液,泳道5为泳道4样品100℃保温30分钟后离心上清液。
具体实施方式
本发明可通过后续对于本发明一些实施方案描述以及其中所包括的实施例的详细内容而更容易被了解。
在进一步叙述本发明之前,应明了本发明不会被局限于所述特定实施方案中,因为这些实施方案必然是多样的。亦应明了本说明书中所使用的用语仅是为了阐述特定实施方案,而非作为限制,因为本发明的范围将会被仅仅界定在所附的权利要求中。
除非本文另有定义,连同本发明使用的科学和技术术语应具有本领域普通技术人员通常理解的含义。术语的含义和范围应当清晰,然而,在任何潜在不明确性的情况下,本文提供的定义优先于任何字典或外来定义。在本申请中,除非另有说明,“或”的使用意味着“和/或”。此外,术语“包含”及其他形式的使用是非限制性的。
为了本发明可以更容易地理解,选择的术语在下文定义。
本发明所述的辅助蛋白为能够与目标蛋白融合表达的蛋白,本发明中的辅助蛋白为硫氧还蛋白,其可以来源于Pyrodictium occultum菌株、Hyperthermus butylicus菌株、Pyrolobus fumarii菌株、Aeropyrum pernix K1菌株,当其为来源于Pyrodictiumoccultum菌株时,对目标蛋白表达分离的效果最优,且与现有技术中其它辅助蛋白相比,稳定性、溶解性及表达效率更优。
本发明中所述的辅助蛋白SEQ ID NO.1-4为本发明人发现的在重组融合蛋白表达效率、溶解性、稳定性上表现突出的蛋白序列,其中SEQ ID NO.1在稳定性方面远优于其它序列,且表达的可溶性蛋白含量高。
本发明中所述的辅助蛋白,在进行密码子优化后,其序列SEQ ID NO.5,与目标蛋白重组后,表达效率最高。
可选的,本发明中所述的辅助蛋白可以为1个或多个密码子优化后的硫氧还蛋白序列。
在一些实施方案中,本发明中所述的辅助蛋白可选的包含在SEQ ID NO.5、SEQ IDNO.6、SEQ ID No.7或SEQ ID NO.8的基础上有1个、2个、3个、4个、5个、6个、7个、8个、9个、10个或多个核苷酸突变的序列。
本发明中所述的连接肽为能够连接两个蛋白模块的柔性多肽,其对于重组重合蛋白的生物活性、稳定性都有一定影响,经发明人实验研究发现,在本发明所述的重组蛋白融合表达系统中,所述的GSG、GSS、GSGSG、GSGGSG、GSGGGS、GSGGSGG均能够提高蛋白表达稳定性、保持目标蛋白的生物活性,并能提高目标蛋白的表达量,其中GSGSG、GSGGSG、GSGGGS效果最优。
本发明中所述的切割位点为辅助蛋白和目标蛋白切割分离的位点,用于将目标蛋白从融合蛋白中分离出来,与蛋白的纯化分离难易程度和纯度相关,经发明人实验研究发现,本发明所述的切割位点中,使用蛋白酶切割位点时切割工具和分离步骤简单、切割效率最高。本发明所述的目标蛋白可以为任意大小,其中在大小为15-100个氨基酸序列时,表达效率较高且可溶性蛋白含量较高,其中表达的目标蛋白可以为胰高血糖素样肽类似物9-37,胰高血糖素样肽-1、降血压肽、血管紧张肽I和II、抗菌肽等及其衍生多肽、促生长激素神经肽、生长激素、促胰液素,但不限于上述蛋白。
本发明中所述的融合表达系统的氨基酸序列为SEQ ID NO.10中所述的氨基酸序列时,表达效率高,所表达的目标蛋白含量最高,可溶性及稳定性最好,分离后目标蛋白纯度最高。
下面将结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限制本发明的范围。以下实施例中试剂均为分析纯试剂,分子克隆相关酶试剂购于New England Biolabs公司,大肠杆菌菌株为本实验室保存,基因合成、引物合成和基因测序由上海生工生物工程有限公司完成。实施例中未详细描述的分子克隆实验步骤均参考《分子克隆实验指南(第四版)》(M.R.格林、J.萨姆布鲁克主编,贺福初主译,北京:科学出版社,2017)。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1:重组融合蛋白表达载体构建
本实施例中构建的融合蛋白表达系统为:辅助蛋白-切割位点-胰高血糖素样肽类似物9-37,氨基酸序列如Seq ID No.8所示,对应的核苷酸序列如Seq ID No.9所示基因。
1.1目标基因的扩增
合成Seq ID No.9中基因,其中辅助蛋白基因为Seq ID No.5所示的硫氧还蛋白核苷酸序列,位于Seq ID No.9所示基因的第1-393位,切割位点基因为肠激酶切割位点基因(DDDDK),位于Seq ID No.9所示基因的第394-408位,所述胰高血糖素样肽类似物9-37基因为Seq ID No.12所示的核苷酸序列,位于Seq ID No.9所示基因的第409-498位。
以含Seq ID No.9基因的合成质粒为模板,利用Seq ID No.14所示正向引物和SeqID No.15所示反向引物(含KpnI酶切位点)扩增融合蛋白基因。PCR程序为98℃热变性10秒,55℃退火温度处理30秒,72℃延伸15秒,重复30个循环。扩增结束后经琼脂糖凝胶电泳回收融合蛋白基因。
1.2启动子基因扩增
以pET-28a质粒为模板,利用Seq ID No.16所示正向引物(含BglII酶切位点)和Seq ID No.17所示反向引物扩增T7启动子序列。PCR反应采用NEB公司的Q5 DNA聚合酶,PCR程序为98℃热变性10秒,58℃退火温度处理30秒,72℃延伸4秒,重复30个循环。扩增结束后经琼脂糖凝胶电泳回收T7启动子基因。
1.3载体连接及扩增
运用SOE-PCR方法连接T7启动子基因和融合蛋白基因,以T7启动子基因和融合蛋白基因为模板,利用Seq ID No.16所示正向引物和Seq ID No.15所示反向引物扩增两基因搭接片段。PCR程序为98℃热变性10秒,58℃退火温度处理30秒,72℃延伸19秒,重复35个。扩增结束后经琼脂糖凝胶电泳回收搭接基因。该搭接片段和pET-28a质粒均用BglII和KpnI限制性内切酶在37℃条件下双酶切1小时,琼脂糖凝胶电泳回收两双酶切片段,然后利用T4DNA连接酶在4℃条件下过夜连接两双酶切片段。利用热激转化法把该连接产物转化至大肠杆菌DH5α感受态细胞中,转化菌液涂布于含50ug/mL硫酸卡那霉素的LB平板上,在37℃培养箱中过夜培养。次日挑取单菌落进行菌落PCR验证,所用验证引物为Seq ID No.16和Seq IDNo.15,正确结果为扩增出约0.5kb大小条带。选取菌落PCR正确的菌株进行质粒测序,测序正确后该质粒则为融合蛋白的表达载体。
实施例2:重组融合蛋白表达载体构建
本实施例中构建的融合蛋白表达系统为:纯化标签-辅助蛋白-连接肽-切割位点-胰高血糖素样肽类似物9-37,氨基酸序列如Seq ID No.10所示,对应的核苷酸序列如SeqID No.11所示基因。
2.1目标基因的扩增
合成Seq ID No.11中基因,其中纯化标签基因为6个组氨酸纯化标签核苷酸序列(HHHHHH),位于Seq ID No.11所示基因的第4-21位,辅助蛋白基因为Seq ID No.5所示的硫氧还蛋白核苷酸序列,位于Seq ID No.11所示基因的第22-414位,切割位点基因为肠激酶切割位点(DDDDK),位于Seq ID No.11所示基因的第430-444位,所述连接肽为GSGSG,位于Seq ID No.11所示基因的第415-429位,所述胰高血糖素样肽类似物9-37基因为Seq IDNo.12所示的核苷酸序列,位于Seq ID No.11所示基因的第445-534位。
以含该基因的合成质粒为模板,利用Seq ID No.18所示正向引物和Seq ID No.15所示反向引物扩增Seq ID No.11基因。PCR程序为98℃热变性10秒,55℃退火温度处理30秒,72℃延伸16秒,重复30个循环。扩增结束后经琼脂糖凝胶电泳回收Seq ID No.11基因。
2.2启动子基因扩增
同实施例1。
2.3载体连接及扩增
将该基因与T7启动子基因进行SOE-PCR形成搭接片段,所用引物对为Seq IDNo.15和Seq ID No.16,PCR程序为98℃热变性10秒,58℃退火温度处理30秒,72℃延伸19秒,重复35个循环。扩增结束后经琼脂糖凝胶电泳回收搭接片段。该搭接片段和pET-28a质粒均用BglII和KpnI限制性内切酶在37℃条件下双酶切1小时,琼脂糖凝胶电泳回收两双酶切片段,然后利用T4 DNA连接酶在4℃条件下过夜连接两双酶切片段。利用热激转化法把该连接产物转化至大肠杆菌DH5α感受态细胞中,转化菌液涂布于含50ug/mL硫酸卡那霉素的LB平板上,在37℃培养箱中过夜培养。次日挑取单菌落进行菌落PCR验证,所用验证引物为Seq ID No.15和Seq ID No.16,正确结果为扩增出约0.55kb大小条带。
实施例3:工程菌株构建和重组融合蛋白制备
3.1工程菌株构建
将实施例2中测序正确的融合蛋白表达载体通过热激转化法导入大肠杆菌BL21(DE3)感受态细胞中,转化菌液涂布于含50ug/mL硫酸卡那霉素的LB平板上,在37℃培养箱中过夜培养。次日挑取单菌落进行菌落PCR验证,验证方法同实施例1。对所得阳性单菌落进行不少于3次的平板分离纯化单菌落,每次皆需进行菌落PCR验证,最终得到的阳性单菌落则为表达融合蛋白的工程菌株。
3.2重组融合蛋白制备
挑取阳性工程菌株单菌落,接种至5mL液体LB培养基(蛋白胨1%,酵母提取物0.5%,NaCl1%),添加终浓度50ug/mL硫酸卡那霉素,37℃、170rpm过夜培养制备种子液。将种子液按1%比例接种至100mL液体LB培养基中(500mL三角瓶),添加终浓度50ug/mL硫酸卡那霉素,37℃、170rpm培养至对数生长期,此时OD600=0.4-0.6,添加终浓度0.2mM异丙基硫代半乳糖苷(IPTG),培养温度调整为20-34℃,170rpm培养,诱导目标蛋白表达。诱导表达18-20小时,摇瓶发酵结束,加热煮沸发酵液并保温30分钟,菌体裂解并释放融合蛋白,大部分宿主蛋白会因加热而变性沉淀,10000хg离心10分钟,收集上清液,融合蛋白存在于上清液中。
配制SDS-PAGE蛋白电泳相关试剂,将上述热裂解离心上清液与蛋白电泳loadingbuffer混合,于100℃沸水中加热5分钟,上样进行SDS-PAGE蛋白电泳,考马斯亮蓝染色后再脱色,蛋白电泳结果如图1所示,泳道1为工程菌热裂解离心上清液蛋白电泳结果,M为蛋白质标准分子量标记。从结果可以看出,热裂解离心上清液中有很明显的融合蛋白条带(17KD),并且只含少量宿主蛋白,说明通过本发明专利的方法可使目标蛋白大量的可溶表达,并且具有热稳定性(100℃耐受至少30分钟),热裂解后杂蛋白基本被去除,裂解液中融合蛋白含量高,纯度也高,有利于简化蛋白质分离纯化工艺,降低纯化成本。通过加热的方式即可获得目标蛋白溶液,不需繁琐低效的超声破碎或高压破碎,不需额外的破碎仪器保养费用,更适合工业化大规模应用。
实施例4:重组融合蛋白的热稳定评价
为了对比辅助蛋白的效果,构建另一个融合蛋白作为阴性对照,阴性对照选择来源于大肠杆菌的硫氧还蛋白作为辅助蛋白,表达载体构建过程和工程菌的制备过程同实施例1。按照实施例3的方法制备热稳定融合蛋白和非热稳定融合蛋白的发酵液,10000хg离心10分钟,收集菌体,用生理盐水重悬两次,用生理盐水调整两样品至相同菌体浓度,超声破碎细胞(功率350W,超声3秒,间歇3秒,工作时间10分钟),10000хg离心10分钟,收集超声破碎上清液。取部分超声破碎上清液于沸水中(100℃)保温30分钟,10000хg离心10分钟,收集加热离心上清液。取超声破碎上清液和加热离心上清液进行SDS-PAGE蛋白电泳,结果如图1所示,其中,M为蛋白质标准分子量标记,泳道2为本发明重组融合蛋白工程菌超声破碎离心上清液,泳道3为泳道2样品100℃保温30分钟后离心上清液,泳道4为阴性对照融合蛋白工程菌超声破碎离心上清液,泳道5为泳道4样品100℃保温30分钟后离心上清液。从结果可以看出,包含本发明表达载体的融合蛋白工程菌超声破碎液中含有大量的宿主蛋白,100℃加热处理后绝大部分宿主蛋白都被除去,而融合蛋白含量几乎没有减少,说明其具有良好的热稳定性(100℃耐受至少30分钟),能够通过简单加热步骤进行纯化处理,包含阴性对照载体的融合蛋白工程菌的超声破碎上清中也有明显的融合蛋白条带,但100℃加热处理后融合蛋白条带消失,说明该硫氧还蛋白可促进蛋白的可溶表达,但却不具备热稳定性。因此,本发明提供的辅助蛋白不仅使融合蛋白可溶表达,减少包涵体形成,还具有高度的稳定性。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,但本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
SEQUENCE LISTING
<110> 丽珠集团新北江制药股份有限公司
<120> 一种辅助蛋白在重组蛋白生产中的应用及融合表达系统
<130> 2020.10.14
<160> 18
<170> PatentIn version 3.5
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130 135 140
Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
145 150 155 160
Val Arg Gly Arg Gly
165
<210> 9
<211> 498
<212> DNA
<213> 人工序列
<400> 9
atgatggaag aggtagactt ttatacacgt atgtatgatg agatcgaacg catgcgtaag 60
gagggtatcg ttgagtacgt gacgagctat gcgcgttttc gtgaaatttt aaaaaatcac 120
cgcgtcacgg tggccgtatt cactacacct acttgcacgg cctgcatggt ctacaaaccg 180
atcttctata tgacggctga gaagctgcgt gatcaggcta agtggattga agtagatgca 240
tacgaagccc ccgaggctgc atttgaatgg ggtgtcacag ctaccccgac taccatcgtt 300
tatgtgaacg gggaagcagt cgaagccttc gtaggggtct tggatgatga atcgttagag 360
gaaatcgtta agggagccgt ggaacgtgtc aaggatgacg atgacaagga aggcaccttt 420
accagcgatg tgagcagcta tctggaaggc caagctgcca aagaatttat tgcttggctg 480
gtgcgcggtc gcggttga 498
<210> 10
<211> 177
<212> PRT
<213> 人工序列
<400> 10
Met His His His His His His Met Met Glu Glu Val Asp Phe Tyr Thr
1 5 10 15
Arg Met Tyr Asp Glu Ile Glu Arg Met Arg Lys Glu Gly Ile Val Glu
20 25 30
Tyr Val Thr Ser Tyr Ala Arg Phe Arg Glu Ile Leu Lys Asn His Arg
35 40 45
Val Thr Val Ala Val Phe Thr Thr Pro Thr Cys Thr Ala Cys Met Val
50 55 60
Tyr Lys Pro Ile Phe Tyr Met Thr Ala Glu Lys Leu Arg Asp Gln Ala
65 70 75 80
Lys Trp Ile Glu Val Asp Ala Tyr Glu Ala Pro Glu Ala Ala Phe Glu
85 90 95
Trp Gly Val Thr Ala Thr Pro Thr Thr Ile Val Tyr Val Asn Gly Glu
100 105 110
Ala Val Glu Ala Phe Val Gly Val Leu Asp Asp Glu Ser Leu Glu Glu
115 120 125
Ile Val Lys Gly Ala Val Glu Arg Val Lys Gly Ser Gly Ser Gly Asp
130 135 140
Asp Asp Asp Lys Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
145 150 155 160
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
165 170 175
Gly
<210> 11
<211> 534
<212> DNA
<213> 人工序列
<400> 11
atgcatcatc atcatcatca catgatggaa gaggtagact tttatacacg tatgtatgat 60
gagatcgaac gcatgcgtaa ggagggtatc gttgagtacg tgacgagcta tgcgcgtttt 120
cgtgaaattt taaaaaatca ccgcgtcacg gtggccgtat tcactacacc tacttgcacg 180
gcctgcatgg tctacaaacc gatcttctat atgacggctg agaagctgcg tgatcaggct 240
aagtggattg aagtagatgc atacgaagcc cccgaggctg catttgaatg gggtgtcaca 300
gctaccccga ctaccatcgt ttatgtgaac ggggaagcag tcgaagcctt cgtaggggtc 360
ttggatgatg aatcgttaga ggaaatcgtt aagggagccg tggaacgtgt caagggctct 420
ggctccggtg atgacgatga caaggaaggc acctttacca gcgatgtgag cagctatctg 480
gaaggccaag ctgccaaaga atttattgct tggctggtgc gcggtcgcgg ttga 534
<210> 12
<211> 29
<212> PRT
<213> 人工序列
<400> 12
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> 13
<211> 90
<212> DNA
<213> 人工序列
<400> 13
gaaggcacct ttaccagcga tgtgagcagc tatctggaag gccaagctgc caaagaattt 60
attgcttggc tggtgcgcgg tcgcggttga 90
<210> 14
<211> 35
<212> DNA
<213> 人工序列
<400> 14
agaaggagat ataccatgat ggaagaagtt gattt 35
<210> 15
<211> 29
<212> DNA
<213> 人工序列
<400> 15
gacggtacct caaccgcgac cgcgcacca 29
<210> 16
<211> 20
<212> DNA
<213> 人工序列
<400> 16
tcgagatctc gatcccgcga 20
<210> 17
<211> 20
<212> DNA
<213> 人工序列
<400> 17
ggtatatctc cttcttaaag 20
<210> 18
<211> 35
<212> DNA
<213> 人工序列
<400> 18
agaaggagat ataccatgca tcatcatcat catca 35
Claims (10)
1.一种辅助蛋白在重组蛋白生产中的应用,其特征在于所述辅助蛋白的氨基酸序列为SEQ ID NO.1、SEQ ID NO.2、SEQ ID No.3或SEQ ID NO.4中的任意一种。
2.根据权利要求1所述的应用,其特征在于所述辅助蛋白的氨基酸序列为SEQ IDNO.1。
3.根据权利要求2所述的应用,其特征在于所述辅助蛋白的核苷酸序列为SEQ IDNO.5、SEQ ID NO.6或SEQ ID No.7中的任意一种;
优选地,所述辅助蛋白的核苷酸序列为SEQ ID NO.5。
4.一种用于重组蛋白生产的融合表达系统,其特征在于包含权利要求1-3中任意一项的辅助蛋白;
优选地,所述融合表达系统包含辅助蛋白-连接肽-切割位点-目标蛋白;
优选地,所述连接肽为GSG、GSS、GSGSG、GSGGSG、GSGGGS、GSGGSGG中的一种,进一步优选地,所述连接肽为GSGSG、GSGGSG或GSGGGS;
优选地,所述连接肽的1个或多个重复,进一步优选地,所述连接肽为1-3个重复;
优选地,所述切割位点为化学切割位点、物理切割位点或蛋白酶切割位点,进一步优选地所述切割位点为蛋白酶切割位点;
更优选地,所述化学切割位点为溴化氰(Met↓)、羟胺(Asn↓Gly)等,物理切割位点为低pH(Asp↓Pro)等,蛋白酶切割位点为Xa因子切割位点、凝血酶切割位点、肠激酶切割位点、烟草蚀纹病毒蛋白酶切割位点、凝乳酶切割位点、胶原酶切割位点、3C蛋白酶切割位点、胰蛋白酶切割位点,进一步优选为肠激酶切割位点。
5.根据权利要求4所述的融合表达系统,其特征在于所述目标蛋白为10-200个氨基酸序列;
优选地所述目标蛋白为15-100个氨基酸序列,更优选地,所述目标蛋白为胰高血糖素样肽-1、降血压肽、血管紧张肽I和II、抗菌肽等及其衍生多肽、促生长激素神经肽,进一步优选地,所述目标蛋白为胰高血糖素样肽-1。
优选地,所述融合表达系统的氨基酸序列为SEQ ID NO.8中所述的蛋白。
优选地,所述融合表达系统的基因编码序列为SEQ ID NO.9中所述的核苷酸序列。
6.根据权利要求4所述的融合表达系统,其特征在于还进一步包含纯化标签,所述纯化标签位于辅助蛋白上游或连接肽和切割位点之间;
优选地,所述纯化标签为亲和纯化标签;
更优选地,所述亲和纯化标签为组氨酸标签、赖氨酸标签、精氨酸标签、谷胱甘肽-S-转移酶标签、FLAG标签,进一步优选为组氨酸标签;
优选地,所述的融合表达系统的氨基酸序列为SEQ ID NO.10中所述的氨基酸序列;
优选地,所述融合表达系统的基因编码序列为SEQ ID NO.11中所述的核苷酸序列。
7.包含权利要求1-6中任意一项所述的辅助蛋白基因或者重组表达系统基因的表达载体;
优选地,所述载体为pET系列、pBAD系列、pTrc系列、pCold系列、pUC系列载体中的任意一种;
优选地,所述载体启动子为T7、lac、Trp、Tac、Trc、λPL、PL、araBAD、GAPDH中的一种或者多种。
8.权利要求1-7中任意一项所述的辅助蛋白或者重组表达系统或载体在生产重组融合蛋白中的应用。
9.一种重组蛋白的制备方法,其步骤为:
1)合成权利要求4-7中任意一项所述的重组表达系统基因;
2)将重组表达系统基因插入载体的多克隆位点,构建包含重组蛋白的表达载体;
3)将重组蛋白表达载体导入表达菌株中,构建重组蛋白表达工程菌;
4)对重组蛋白表达工程菌进行发酵,表达重组蛋白;
5)加热处理发酵液,使工程菌裂解释放重组蛋白,过滤去除沉淀,收集破碎上清液,得到重组蛋白。
优选地,所述加热温度为60-100℃,进一步优选地,所述加热温度为100℃。
优选地,所述加热时间为30-60分钟,进一步优选地,所述加热时间为30分钟。
10.根据权利要求9所述的制备方法,其特征在于,所述表达菌株为原核表达菌株,优选为大肠杆菌。
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CN110564797A (zh) * | 2019-08-14 | 2019-12-13 | 成都英普博集生物科技有限公司 | 一种利用热稳定融合蛋白制备多肽的方法 |
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CN110564797A (zh) * | 2019-08-14 | 2019-12-13 | 成都英普博集生物科技有限公司 | 一种利用热稳定融合蛋白制备多肽的方法 |
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