CN110655584A - 自断蛋白的活性控制及其应用 - Google Patents
自断蛋白的活性控制及其应用 Download PDFInfo
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- CN110655584A CN110655584A CN201811192882.0A CN201811192882A CN110655584A CN 110655584 A CN110655584 A CN 110655584A CN 201811192882 A CN201811192882 A CN 201811192882A CN 110655584 A CN110655584 A CN 110655584A
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Abstract
本发明提供一种在无酵素参与下,具自断能力的自断蛋白,以及如何控制该自断蛋白来生产目标蛋白的方法。自断蛋白主体为一内含蛋白,所述内含蛋白的N端氨基酸包含至少有一突变,本发明的自断蛋白在“抑制条件”下,自断蛋白不会产生自我断裂,在“促进条件”下,自断蛋白的C‑端会产生自我断裂,而N‑端则不会产生自我断裂。本发明另提供一种含有该自断蛋白及目标蛋白的融合蛋白。
Description
【技术领域】
本发明是有关于一种自断蛋白,包含此自断蛋白的融合蛋白,以及控制自断蛋白生产目标蛋白的方法。
【先前技术】
内含蛋白是一种天然存在的自我剪接蛋白结构域,功能为从较大的蛋白质结构中自我切除,同时将两侧的胜肽“外显子(exteins)”连接在一起形成成熟的宿主蛋白。
内含蛋白因可重新排列两侧的键结,造就出许多使用内含蛋白的生物技术。这些技术包括各种类型的蛋白质连接、活化,以及蛋白质标记和追踪应用。内含蛋白还有一个重要应用是生产、纯化重组蛋白。特别是,内含蛋白具有自我切除的能力,因此可为研究、医疗及其他商业上应用做出重大的应用。
传统的标签序列为纯化目标蛋白提供了简单而有力的方式,可利用简单的基因重组技术与目标蛋白融合。这些标签已广泛地使用于现在的研究中,并且成为研发与制造的主要平台。然而,虽然目标蛋白可在适当的宿主细胞中表现,并利用标签序列纯化,但标签序列的存在可能会降低目标蛋白的活性,并可能产生不需要的免疫原性。因此,在纯化后将亲和性标签去除是非常重要的,一般是利用高特异性的内切酶进行切除。虽然这些酵素通常有效,但是酵素过于昂贵,对于量产不利,且需要额外的步骤来去除这些酵素。
因此,内含蛋白提供一般标签自我切割的能力是一项重大进步,已有一些文献揭示使用内含蛋白的自我切割能力来移除亲和性标签序列的系统。然而,此方法仍然存在一些重大缺点,以阻碍内含蛋白的应用。例如,在蛋白质表现及纯化的过程必须确实抑制内含蛋白的自我切割反应,但一旦要纯化目标蛋白,则需要控制反应能快速完成;在常见的内含蛋白中,经常需要额外添加硫醇化合物(如,二硫苏糖醇)来诱发断裂,增加成本及事后移除的程序。
因此,业界需要一种稳定、不需额外填加酵素或硫醇化合物、低成本的蛋白质纯化的方法。
【发明内容】
有鉴于此,本发明提供一种自断蛋白、包含此自断蛋白的融合蛋白,以及利用此自断蛋白生产目标蛋白的方法。
本发明提供一种自断蛋白,包括内含蛋白,其中所述的内含蛋白的N-端氨基酸至少包含一个突变,移除N-端自我断裂活性,只保留C-端自我断裂活性,通过调控盐浓度、pH值和温度控制自断蛋白的活性。
在本发明实施例中,所述内含蛋白的N端突变,氨基酸经置换、删除、插入及/或取代。
在本发明实施例中,所述内含蛋白源自于Npu DnaE。
在本发明实施例中,所述内含蛋白N端第1个氨基酸由半胱氨酸(Cys)置换为甘氨酸(Gly)或丙氨酸(Ala)。
在本发明实施例中,所述自断蛋白的序列由下列族群组成:SEQ ID NO:1或/和SEQID NO:2。
在本发明实施例中,所述自断蛋白的序列为:
GLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPNIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN
在本发明实施例中,所述自断蛋白的序列为:
ALSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPNIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN
本发明另提供一种融合蛋白,具有下列式(I)结构:
(X)n-(I)-(P)m (I)
其中,
X为氨基酸序列、连接子、磁珠或其类似物,
I为上述的自断蛋白,
P为目标蛋白,
n为0以上的整数,以及
m为1以上的整数。
在本发明实施例中,所述X为标签序列,此标签序列包括His-Tag、GST-Tag,或其他可供协助纯化及表现的氨基酸序列。较佳的标签序列為有利于在pH值中性至酸性条件下收集、纯化该融合蛋白。
在本发明实施例中,此目标蛋白P包括多肽、蛋白质、重组蛋白、抗体、酵素或激素。
本发明另提供一种蛋白质表达载体,包含本发明所述自断蛋白的核苷酸序列。
在本发明实施例中,此蛋白质表达载体包括His-Tag、GST-Tag或其他可供协助纯化及表现的氨基酸的核苷酸序列。较佳的标签序列為有利于在pH值中性至酸性条件下收集、纯化该融合蛋白。
在本发明实施例中,本发明的自断蛋白的核苷酸序列由下列族群组成:SEQ IDNO:3或/和SEQ ID NO:4。
在本发明实施例中,此自断蛋白的核苷酸序列为:
GGCCTGAGCTATGAAACCGAAATTCTGACCGTGGAATATGGCCTGCTGCCGATTGGTAAAATTGTGGAAAAACGCATCGAATGTACTGTTTATAGCGTTGATAATAATGGCAATATTTATACCCAGCCGGTGGCACAGTGGCACGATCGCGGCGAACAGGAAGTGTTTGAATATTGTCTGGAAGATGGTAGCCTGATTCGCGCAACCAAAGACCATAAATTTATGACTGTTGATGGTCAGATGCTGCCGATTGATGAAATTTTTGAACGTGAACTGGATCTGATGCGCGTTGATAATCTGCCGAATATCAAAATTGCGACCCGTAAATATCTGGGCAAACAGAATGTGTATGACATTGGCGTTGAACGCGACCATAATTTTGCGCTGAAAAATGGCTTCATTGCTTCTAAT
在本发明实施例中,此自断蛋白的核苷酸序列为:
GCGCTGAGCTATGAAACCGAAATTCTGACCGTGGAATATGGCCTGCTGCCGATTGGTAAAATTGTGGAAAAACGCATCGAATGTACTGTTTATAGCGTTGATAATAATGGCAATATTTATACCCAGCCGGTGGCACAGTGGCACGATCGCGGCGAACAGGAAGTGTTTGAATATTGTCTGGAAGATGGTAGCCTGATTCGCGCAACCAAAGACCATAAATTTATGACTGTTGATGGTCAGATGCTGCCGATTGATGAAATTTTTGAACGTGAACTGGATCTGATGCGCGTTGATAATCTGCCGAATATCAAAATTGCGACCCGTAAATATCTGGGCAAACAGAATGTGTATGACATTGGCGTTGAACGCGACCATAATTTTGCGCTGAAAAATGGCTTCATTGCTTCTAAT
本发明另提供一种生产、纯化蛋白质的方法,包括:
(a)在宿主中表达上述融合蛋白;
(b)在抑制条件下收集、纯化所述融合蛋白;
(c)将所述融合蛋白置于促进条件,使所述融合蛋白中的自断蛋白自C-端断裂,且目标蛋白与所述自断蛋白分离;以及
(d)收集、纯化所述目标蛋白。
在本发明实施例中,所述抑制条件为pH值中性至酸性条件。
在本发明实施例中,所述抑制条件为高盐度环境。
在本发明实施例中,所述抑制条件为pH值4-7、盐浓度300mM以上及/或温度为25℃以下。
在本发明实施例中,所述促进条件为pH碱性条件。
在本发明实施例中,所述促进条件为低盐度环境。
在本发明实施例中,所述促进条件为pH值7-11、盐浓度300mM以下及/或温度为25℃以上。
本发明所使用的“盐类”,包含氯化钠及其他水溶性带电离子。
【附图说明】
图1为本发明融合蛋白的结构示意图。图1显示融合蛋白在特定环境下会断裂。
图2显示本发明中实施例中所使用的大肠杆菌表达载体pET-NpuDnaEC1GTag。
图3显示本发明中实施例NpuDnaE-Mms6融合蛋白的SDS-PAGE分析结果,其中Mms6为目标蛋白。利用SDS-PAGE分析融合蛋白从表现至纯化过程中,各步骤中的NpuDnaE-Mms6融合蛋白。
图4显示本发明中实施例NpuDnaE-Mms6融合蛋白断裂前、后的SDS-PAGE分析结果。在本发明NpuDnaE-Mms6融合蛋白断裂后,NpuDnaE融合蛋白断裂成2个蛋白片段。
图5显示Mms6目标蛋白的MALDI-TOF MS质谱仪分析结果。以MALDI-TOF MS质谱仪分析由NpuDnaE-Mms6融合蛋白所分离出的Mms6蛋白,分析结果显示与天然Mms6蛋白具有相同的分子量。
【实施方式】
本发明提供一种自断蛋白。本发明的自断蛋白为内含蛋白,所述内含蛋白的N-端氨基酸有至少一突变,来移除N-端自我断裂活性,只保留C-端自我断裂活性,通过调控盐浓度、pH值和温度来控制自断蛋白的活性。
本发明所述的N-端氨基酸至少包含一个“突变”,即为“修饰的”或“突变的”内含蛋白,是指在内含蛋白的N-端氨基酸序列上,相较于天然序列或正常结构有一或多个改变。此改变可为置换、添加、修饰或删除。
本发明所述的氨基酸的置换、添加、修饰或删除可以是天然或非天然氨基酸。天然氨基酸包括丙氨酸、精氨酸、天冬酰胺、天冬氨酸、半胱氨酸、谷氨酸、谷氨酰胺、甘氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸及缬氨酸。非天然氨基酸包括,但不限于,ε-N赖氨酸、β-丙氨酸、鸟氨酸、正亮氨酸、正缬氨酸、羟脯氨酸、甲状腺素、γ-氨基丁酸、丝氨酸、瓜氨酸、氨基苯甲酸、6-氨基己酸(Aca;6-Aminohexanoic acid)、羟脯氨酸、巯基丙酸(MPA)、3-硝基-酪氨酸、焦谷氨酸及其类似物。
在本发明实施例中,本发明自断蛋白可源自于NpuDnaE,其N-端的第1个氨基酸由半胱氨酸(Cys)置换为其他氨基酸,较佳置换为甘氨酸(Gly)或丙氨酸(Ala)等,但不得为丝氨酸(Ser)或苏氨酸(Thr)。因此,突变后内含蛋白的N-端不具有断裂活性,但C-端仍具有断裂活性。本发明自断蛋白可具有SEQ ID NO:1或/和SEQ ID NO:2的序列。
在一实施例中,本发明的自断蛋白在“抑制条件”下,自断蛋白的N-端及C-端的断裂活性皆被抑制。本发明的“抑制条件”为pH值中性至酸性的条件,较佳的pH值为4至7;盐浓度为高盐度环境,较佳的盐浓度为300mM以上;且较佳的抑制条件之温度为25℃以下。
在一实施例中,本发明的自断蛋白在“促进条件”下,自断蛋白的C-端断裂活性被活化。由于本发明的自断蛋白的N-端氨基酸经突变、修饰过,已失去断裂活性,故通常仅C-端会产生断裂。
本发明的“促进条件”为pH值为碱性的条件,较佳的pH值为7至11;盐浓度为低盐度环境,较佳的盐浓度为300mM以下;且较佳的促进条件的温度为25℃以上。
本发明所使用的“盐类”,不限于氯化钠,亦包含其他水溶性带电离子。
本发明的融合蛋白包括本发明的自断蛋白,在自断蛋白的N-端连接标签序列、连接符、磁珠或其类似物,而自断蛋白的C-端连接目标蛋白。
本发明所述的“标签序列”是指可专一性与一特定配体(ligand)反应的胜肽(肽)。为了进行亲和性纯化,目标蛋白经常与一可与配体专一性结合的标签序列结合。各种已开发的标签包括,但不限于,myc tag、Flag-peptide tag、His-Tag、Strep-Tag、GST-Tag、MBP-Tag、SNAP-Tag、Halo-Tag、Tap-Tag及INPACT-CN。较佳的标签序列為有利于在pH值中性至酸性条件下收集、纯化该融合蛋白。
本发明所示“目标蛋白”是指一生物分子,例如蛋白质及重组多肽等。
由上述可知,本发明自断蛋白的N-端包含有一或多个突变,导致N-端不具有断裂活性。在本发明所述的融合蛋白中,自断蛋白与目标蛋白可选择性地在一特定环境下产生断裂或维持键结连接。如图1,融合蛋白具有标签序列、自断蛋白及目标蛋白。由宿主所产生的融合蛋白在抑制条件下(如,pH值中性至酸性,高盐度),融合蛋白不会产断裂现象。然而,将融合蛋白置于促进条件下(如,pH值碱性,低盐度),则自断蛋白的C-端断裂,目标蛋白与自断蛋白分离。可进一步使用已知的分离、纯化方法,获得目标蛋白。
本发明另提供一种生产、制备分离蛋白的方法,包括(a)在宿主中表达本发明所述的融合蛋白,(b)在抑制条件下收集、纯化所述融合蛋白,(c)将融合蛋白置于促进条件,使所述融合蛋白中的自断蛋白产生断裂,且目标蛋白与所述自断蛋白分离,(d)收集、纯化该目标蛋白。
NpuDnaE为断裂内含肽。
利用基因分子工程先行构筑表现(express,表达)融合蛋白的载体,并将载体导入宿主中进行表达。本发明的融合蛋白及表达载体包含于动物细胞、低等真核生物或原核生物各式表现系统。动物细胞可包括猴COS细胞、CHO细胞、人类肾脏293细胞、人类表皮A431细胞、人类Colo205细胞、3T3细胞、CV-1细胞、其他经转殖的灵长类细胞、正常的双套细胞、由体外培养的原生组织衍生细胞株、原生移殖体、HeLa细胞、老鼠L细胞、BHK、HL-60、U937、HaK或Jurkat细胞。低等真核生物包括酵母菌,如Saccharomyces cerevisiae、Schizosaccharomyces pombe、Kluyveromyces strains、Candida或任何可表现异源性蛋白的酵母菌。原核生物可包括Escherichia coli、Bacillus subtilis、Salmonellatyphimurium、或任何可表现异源性蛋白的细菌。
本发明另提供表达载体,其中包含前述的自断蛋白的核苷酸序列。
本发明所述的“载体”包含但不限于pET、pQE、pICZa、pFastBac、pGFP2,或任何可表现异源性蛋白的载体。
在一实施例中,本发明的表达载体包含SEQ ID NO:3或/和SEQ ID NO:4的序列。
在一实施例中,本发明的表达载体包括His-Tag或GST-Tag的核苷酸序列,或其他已开发的标签序列的氨基酸序列。较佳的标签序列為有利于在pH值中性至酸性条件下收集、纯化所述融合蛋白。
在融合蛋白表现、收集及纯化时,应将环境控制在适合宿主生长且不会造成蛋白产生断裂的条件下。在一实施例中,可将融合蛋白置于本发明的“抑制条件”下,进行融合蛋白的表现、收集与纯化。
接着,将纯化的融合蛋白置于本发明的“促进条件”下,使自断蛋白产生断裂,目标蛋白与自断蛋白分离(图1)。最后,收集、纯化所述的目标蛋白。
本发明方法具有以下优点:(1)只需控制水溶液条件,无需酵素或其他额外物质,节省生产成本;(2)本发明所使用的自断蛋白(内含蛋白)对其后方所连接的序列有广泛的容忍性;以及(3)不会有任何残留序列于C端后的目标蛋白上。
实施例1
融合蛋白的制备:构筑编码NpuDnaEC1G融合蛋白(含有His-tag标签、NpuDnaEC1G及Mms6蛋白)于表达载体pET-NpuDnaEC1GTag上(如图2),将表达载体导入E.coli中,加入IPTG诱导NpuDnaE融合蛋白的表达,将NpuDnaE融合蛋白置于高盐环境(300mM NaCl)并经镍离子树酯纯化后,以SDS-PAGE分析(图3)。
由图3可知,在加入IPTG诱导蛋白表达后,E.coli可大量表达NpuDnaE融合蛋白。NpuDnaE融合蛋白主要存在于上清液中,且可经镍离子树酯纯化。NpuDnaE融合蛋白在高盐环境(300mM NaCl以上),不会断裂。
实施例2
融合蛋白的断裂:利用缓冲液交换将NpuDnaE融合蛋白溶液的盐浓度降至300mMNaCl以下,pH值增加至碱性环境,置于50℃下,反应30分钟。将NpuDnaE融合蛋白以SDS-PAGE分析(图3)。由图4可知,NpuDnaE融合蛋白裂解成2个片断,分别是分子量20kDa的NpuDnaE融合蛋白(含有His-tag标签及NpuDnaE内含蛋白),以及17kDa的NpuDnaE自断蛋白。
实施例3
质谱分析:利用高效液相层析法(HPLC)分离实施例2中的NpuDnaE自断蛋白与Mms6目标蛋白,并利用MALDI-TOF MS质谱仪分析Mms6蛋白(第5图)。由图4所示,测得Mms6蛋白分子量为2492.23kDa,而Mms6蛋白分子量的理论值为2492.7kDa,两者几乎相同。
序列表
<110> 台湾国立清华大学
<120> 自断蛋白的活性控制及其应用
<130> THPA-2018001
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Claims (20)
1.一种自断蛋白,包括一内含蛋白,其特征是,所述内含蛋白的N端第1个氨基酸有一突变,其中所述内含蛋白源自于NpuDnaE。
2.如权利要求1所述的自断蛋白,其特征是,所述突变包括置换、删除、插入及/或取代。
3.如权利要求1所述的自断蛋白,其特征是,所述内含蛋白N端第1个氨基酸半胱氨酸(Cys)经由所述突变而改变N端序列,造成所述内含蛋白失去N-端自我断裂活性。
4.如权利要求1所述的自断蛋白,其特征是,所述自断蛋白的序列系包括:SEQ ID NO:1或SEQ ID NO:2。
5.一种融合蛋白,具有下式(I)结构:
(X)n-(I)-(P)m (I)
其中,
X为氨基酸序列、连接符、磁珠或其类似物;
I为权利要求1所述的自断蛋白;
P为目标蛋白;
n为0以上的整数;以及
m为1以上的整数。
6.如权利要求5所述的融合蛋白,其特征是,所述X为一标签序列,所述标签序列包括任何供协助纯化及表达的氨基酸序列。
7.如权利要求6所述的融合蛋白,其特征是,所述标签序列包括但不限于His-Tag或GST-Tag。
8.如权利要求5所述的融合蛋白,其特征是,所述目标蛋白为多肽、蛋白质、重组蛋白、抗体、酵素或激素。
9.一种蛋白质表达载体,其特征是,所述蛋白质表达载体包含如权利要求1所述的自断蛋白的核苷酸序列。
10.如权利要求9所述的蛋白质表达载体,其特征是,所述核苷酸序列包括:SEQ ID NO:3或SEQ ID NO:4。
11.如权利要求9所述的蛋白质表达载体,其特征是,所述包括但不限于His-Tag或GST-Tag的核苷酸序列。
12.一种生产、纯化蛋白质的方法,包括:
(a)在宿主中表达如权利要求5所述的融合蛋白;
(b)在抑制条件下收集、纯化所述融合蛋白,其中所述抑制条件为pH值中性至酸性条件;
(c)将所述融合蛋白置于促进条件,使所述融合蛋白中的自断蛋白断裂,且目标蛋白与所述自断蛋白分离,所述促进条件为pH碱性条件;以及
(d)收集、纯化该目标蛋白。
13.如权利要求12所述的方法,其特征是,所述pH值中性至酸性条件为pH值4-7。
14.如权利要求12所述的方法,其特征是,所述抑制条件为高盐度环境。
15.如权利要求14所述的方法,其特征是,所述高盐度环境为盐浓度300mM以上。
16.如权利要求12所述的方法,其特征是,所述抑制条件为温度为25℃以下的环境。
17.如权利要求12所述的方法,其特征是,所述pH碱性条件为pH值7-11。
18.如权利要求12所述的方法,其特征是,所述促进条件为低盐度环境。
19.如权利要求18所述的方法,其特征是,所述低盐度环境为盐浓度300mM以下。
20.如权利要求12所述的方法,其特征是,所述促进条件为温度为25℃以上的环境。
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