CN114133458B - 一种在人血清白蛋白内部融合多肽的方法 - Google Patents
一种在人血清白蛋白内部融合多肽的方法 Download PDFInfo
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Abstract
本发明公开一种在人血清白蛋白内部融合多肽的方法。本发明所述方法是以HSA的晶体结构为基础，选择在HSA的两个转弯C168‑C177与C360‑C369上，通过基因融合插入具有生物活性的多肽；所选择的两个转弯位于HSA三级结构表面且向外凸，结构紧凑，因此该方法不影响HSA的二级结构及药物结合能力，而且能够维持多肽的高生物活性。在生物工程制药业及基因工程、生物化学、分子生物学等研究领域具有重要应用价值。
Description
技术领域
本发明属于生物医药技术领域，具体涉及一种在人血清白蛋白内部融合多肽的方法。
背景技术
人血清白蛋白 (human serum albumin, HSA) 是血浆的主要成分，在血浆中的浓度为35 ~ 50 mg/ml。HSA由 585 个氨基酸组成，其相对分子质量约为 66.5kDa，正常情况下不易透过肾小球，故半衰期长达19天。HSA既可以通过化学结合也可以通过基因融合有效延长目的蛋白或多肽的半衰期，是一种理想的生物活性蛋白或多肽的载体。
HSA融合技术是指利用基因工程技术，将目的蛋白基因与 HSA 基因融合后，选择合适的系统进行表达，最终获得半衰期更长的药物，已经发展成为蛋白质药物长效化改造的一个通用技术，近年来在长效干扰素、粒细胞生长因子以及白介素等药物的研制上得到了广泛的应用。通常，多肽都是融合在HSA的N端或C端，然而，这种首尾相连的融合方式往往会降低多肽的活性。据报道，将胰高血糖素样肽-1受体 (GLP-1R)的一个多肽激动剂AGLP-1融合在HSA的N端有效延长了其半衰期，然而相比于AGLP-1，融合蛋白与GLP-1R的结合能力降低了100倍。同样地，生长抑素-14 (somatostatin-14, SST-14)对所有生长抑素受体亚型(somatostatin receptor subtypes, SSTRS)都具有高亲和活性。然而，将两段重复的SST-14融合在HSA的N端，所得到的融合蛋白(SST14)2-HSA对所有受体亚型的亲和活性都降低了。相比于SST-14，(SST14)2-HSA 对5种SSTR的亲和力都表现出不同程度的下降。
因此，为了维持多肽的高生物活性，构建多肽与HSA的融合蛋白时，合适的融合位点非常重要。
发明内容
本发明的目的在于针对上述问题，提供一种在人血清白蛋白（HSA）内部融合多肽的方法。该方法基于HSA的晶体结构，找到新的适合融合多肽的位点，即两段位于HSA三级结构表面的两个转弯 (C168-C177与C360-C369) 上，提供一种在HSA内部融合多肽且维持多肽高活性的融合方法。
为实现上述目的，本发明采用如下技术方案：
一种在人血清白蛋白（HSA）内部融合多肽的方法：将具有生物活性的多肽融合在人血清白蛋白(human serum albumin, HSA)结构域Ⅰ和Ⅱ的两条α螺旋链之间两段转弯C168-C177与C360-C369 位点上。
上述两段转弯 C168-C177与C360-C369 位点，附近存在两对二硫键 C168-C177和C169-C124 ； C360-C369和C361-C316；结构紧凑，有利于维持多肽的活性构象；且该两段转弯位于HSA表面并向外凸出，有利于消除HSA与多肽之间以及融合蛋白与靶向受体之间的空间位阻；该两段转弯远离HSA与新生儿Fc受体(FcRn)的结合位点，有利于保持HSA长达19天半衰期的特性，改善融合多肽的药代动力学性质。
上述具有生物活性的多肽包括但不限于：氨基酸序列为CVSNKYFSNIHWC多肽；或氨基酸序列为CPAYSRYIGC的多肽。
一种在人血清白蛋白（HSA）内部融合多肽的方法，包括以下步骤：
（1）内部融合位点选择：内部融合位点选择位于HSA结构域Ⅰ和Ⅱ的两条α螺旋链之间两段转弯C168-C177与C360-C369上；
（2）内部融合蛋白的构建和表达：多肽编码基因融合到HSA两段转弯C168-C177与C360-C369的基因位置上，并在真核表达系统中进行高效表达。
上述方法中多肽编码基因融合的方法包括但不限于PCR或LIC方法。
上述方法中所述真核表达系统包括但不限于：酵母表达系统。
一种在人血清白蛋白（HSA）内部融合多肽的方法在生物医药方面的应用。
本发明的优点在于：提供一种在HSA内部融合多肽的方法。该方法所得到的融合蛋白产率高、纯度高、能够维持HSA的二级结构及药物结合能力。而且相比于将多肽融合在HSA的N端，该方法能够维持多肽的高活性。本发明将HSA内部融合作为一个通用和有效的方法，用于生物活性多肽的融合表达，保持多肽生物学活性的同时，不影响HSA的结构与功能，适用于生物工程制药业及基因工程、生物化学、分子生物学等研究领域。
附图说明
图1：HSA晶体结构图。HSA由3个结构域组成(I，Ⅱ，Ⅲ)，两个用于融合多肽的小转弯分别位于结构域Ⅰ和结构域Ⅱ，并向外凸出，附近存在两对二硫键，结构紧凑。
图2：HSA-Ω与Ω-HSA载体的构建及蛋白的表达与纯化。A和B分别为HSA-Ω与Ω-HSA载体构建示意图，C为蛋白纯化后的SDS-PAGE电泳图。
图3：圆二色谱测定HSA-Ω与HSA的二级结构。
图4：荧光探针表征HSA-Ω与HSA上两个特异性药物结合位点。A为丹酰胺特异性结合药物结合位点Ⅰ，B为丹磺酰甘氨酸特异性结合药物结合位点Ⅱ 。
图5：荧光偏振测定HSA-Ω及Ω-HSA与suPAR的结合。
图6：所述融合方法适用于另一多肽IG2。A为HSA-IG2与IG2-HSA蛋白纯化后的SDS-PAGE电泳图，B为圆二色谱测定HSA-IG2与HSA的二级结构，C为HSA-IG2与IG2-HSA抑制muPA水解S-2444能力的测定。
具体实施方式
下面将结合附图和实施例对本发明的方法和其优点作进一步说明，但这些实施例并不构成对本发明权利要求范围的限制。在不脱离本发明主要特征的范围下，本发明还会有各种变化和改进，这些变化和改进均落入本发明保护的范围内。
实施例一：HSA与uPA19-31的融合
uPA19-31是一段来源于尿激酶型纤溶酶原激活剂 (urokinase type plasminogenactivator, uPA) 氨基末端的肽链，与可溶性尿激酶受体 (soluble urokinasereceptor, suPAR) 具有高亲和力 (流式细胞术测得二者结合的IC50值约为700 nM)，其氨基酸序列为CVSNKYFSNIHWC，将其命名为Ω。本实施例中，基于HSA的晶体结构(图1)，将Ω两端的半胱氨酸去除后融合在HSA转弯位置C360-C369上(以验证该转弯是否能够对Ω形成足够的构象限制)的A363与A364之间，得到融合蛋白HSA-Ω（氨基酸序列如SEQ ID NO.1所示）。作为对照，将Ω融合在HSA的N端(Ω的C端与HSA的N端连接，二者之间加入一段连接序列GGGGSGGGGSGGGGS以消除潜在的空间位阻)，得到Ω-HSA。
(1)融合表达载体的构建。
重组融合蛋白表达载体的构建采用不依赖连接反应的克隆(Ligation-Independent Cloning, LIC)方法，示意图如图2A所示。以pPICZα-HSA(构建方法参考“朱丽丽. 人血清白蛋白的表达和结构研究以及蛇毒金属蛋白酶Jerdonitin的研究[D]. 福建:中国科学院福建物质结构研究所,2008.”)质粒为模板，使用引物：5’-GCAGATCCTCATGAATGCTATGCCA-3’ (上游引物), 5’-AGCGGCACAGCACTTCTCTAGA-3’ (下游引物)， PCR扩增得到A363和A364之间断开的线性化载体。PCR产物经核酸外切酶III酶切，产生缺口用以插入编码VSNKYFSNIHW且含有与载体具有15bp重叠的引物对（上游引物为5’-AAGTGCTGTGCCGCTGTGTCCAACAAGTACTTCTCCAACATTCACTGG-3’， 下游引物为5’-TTCATGAGGAT CTGCCCAGTGAATGTTGGAGAAGTACTTGTTGGACAC-3’，划线部分为编码融合多肽的序列）。引物对在100℃下加热5min，自然冷却至室温，形成具有粘性末端的双链，作为LIC的插入物。酶切后的载体和退火后的引物对混合后，65℃加热5min，冰浴5min，转化至DH5α感受态，于含有30μg/ml Zeocin®的LLB培养皿上筛选得到阳性菌落，DNA测序以验证HSA-Ω表达载体的构建，成功构建重组表达质粒pPICZα-HSA-Ω。Ω-HSA表达载体的构建采用相同的策略，用于扩增载体的引物为：5’-GCGGCCGCAGGTGGTGGTGGTTC-3’(上游引物)，5’-GAATTCAGCTTCAGCCTCTCTTTTCTCGAGAGATACCCC-3’(下游引物)，编码目的片段，用于融合在N端的引物对为：上游引物为5’-GCTGAAGCTGAATTCTGTGTGTCCAACAAGTACTTCTCCAACATTCACTGGTGC-3’，下游引物为5’-ACCACCTGCGGCCGCGCACCAGTGAATGTTGGAGAAGTACTTGTTGGACACACA-3’，划线部分为编码融合多肽的序列；成功构建重组表达质粒pPICZα-Ω-HSA。
(2)蛋白的表达与纯化。
将pPICZα-HSA质粒、重组表达质粒pPICZα-HSA-Ω或pPICZα-Ω-HSA经PmeI酶线性化后，电转至毕赤酵母菌株X-33中进行小量表达。将小量表达成功X-33菌株在1 L的培养体系中进行大量表达，收集上清按以下步骤进行纯化：①取上清抽滤液加入预先平衡的 Ni-NTA 层析柱 (GE公司，流速5 mL/min，柱体积25 mL) ；②2倍柱体积平衡液 (20 mMTris-HCl pH 7.4，500 mM NaCl)平衡Ni-NTA 柱；③3倍柱体积去杂蛋白溶液(20 mM Tris-HClpH 7.4，500 mM NaCl，5 mM咪唑)对Ni-NTA 柱进行冲洗，并用紫外检测仪进行检测，直至检测到蛋白质吸收光谱消失；④用去杂液(20 mM Tris-HCl pH 7. 4，体积分数为30%的酒精，6 M尿素) 对结合有目的蛋白的Ni-NTA柱进行10倍柱体积冲洗；⑤用平衡液 ( 20 mMTris-HCl pH 7. 4，500 mM NaCl) 对 Ni-NTA柱进行6～8倍柱体积冲洗，使蛋白复性；⑥蛋白洗脱液 ( 20 Mm Tris-HCl pH 7. 4，500 mM NaCl，500 mM咪唑) 对结合在 Ni-NTA 填料上的蛋白进行洗脱，整个过程用紫外检测仪检测，并进行蛋白峰收集。蛋白纯度通过12%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳进行确定，结果(图2B)表明，HSA-Ω与Ω-HSA均能在酵母系统中高表达，经上述纯化步骤后，蛋白纯度可达99%以上，与HSA一致。
(3)圆二色谱(CD)测定HSA-Ω与HSA的二级结构
用JASCO J1500分光光度计采集HSA-Ω 与HSA的圆二色谱。蛋白浓度为1 μM，光谱测量范围为190 ~ 260 nm，步长为0.1 nm，带宽为1 nm，响应时间为1 s。每个图谱代表了连续三次扫描图和数据分析的平均值。平均椭圆率MRE = CD (mdeg) /(10cnl), c是蛋白质的摩尔浓度, n是氨基酸残基的数量(HSA- Ω为596，HSA为585)，l为路径长度(0.1 cm)。α-螺旋(%)= [(- MRE209 - 4000) / (33000 - 4000)] × 100。结果(图 3)表明，HSA-Ω与HSA的圆二色谱峰型一致，多肽的插入不影响HSA的二级结构。
(4)HSA上两个特异性药物结合位点的表征
HSA的两个主要药物结合位点为药物结合位点Ⅰ(位于结构域ⅡA)和位点Ⅱ(位于结构域ⅢB)（见图1），通过比较HSA-Ω和HSA结合两个荧光探针(丹酰胺和丹磺酰甘氨酸分别与位点Ⅰ和Ⅱ特异性结合, 结合后荧光强度显著增强)的能力，探究多肽的融合对HSA结合药物的能力是否产生影响。固定浓度的荧光探针(丹酰胺和丹磺酰甘氨酸，终浓度分别为40 nM和10 nM）和三倍梯度稀释（稀释缓冲液为20 mM Tris-HCl pH 7.4, 150 mM NaCl,1%DMSO) 的HSA-Ω 或 HSA依次加入384孔黑板中，室温孵育30min。酶标仪测定荧光强度( λex =365 nm，λem = 480 nm )，通过相对荧光值(RFU)对LogC进行非线性拟合确定IC50值(图4)。结果显示，HSA-Ω与HSA对两个荧光探针的结合能力相当，表明插入融合的多肽不影响HSA的药物结合能力。
(5)荧光偏振法测定HSA-Ω及Ω-HSA与suPAR的结合
采用荧光偏振法测定HSA-Ω与Ω-HSA竞争性结合suPAR的能力，其原理为: 荧光小分子5-羧基荧光素(5-FAM)标记的suPAR靶向肽AE147 (记为AE147-FAM，由浙江鸿拓公司合成)与受体suPAR结合后荧光偏振值增强，加入配体蛋白后，配体与AE147-FAM竞争性结合受体，荧光偏振值减弱。固定浓度的suPAR和AE147-FAM (终浓度均为50 nM, 缓冲液为20mMTris-HCl pH 7. 4, 50mM NaCl) 分别加到96孔黑板中，37℃孵育30min后加入三倍梯度稀释的配体蛋白（Ω、HSA-Ω及Ω-HSA）溶液(稀释溶液为 20mM Tris-HCl pH 7. 4, 50mMNaCl), 室温孵育1 h后，用酶标仪进行荧光偏振测定(λex =495 nm, λem = 535 nm), 荧光偏振值(mp)对LogC进行非线性拟合，得到蛋白与荧光标记肽竞争性结合suPAR的IC50值。如图5所示，HSA-Ω与suPAR具有高亲和活性，与荧光标记靶向肽AE147 竞争结合suPAR的IC50值为37 nM，比游离的Ω多肽亲和力更强(IC50值为950 nM)，而Ω-HSA不显示与suPAR结合的活性。
(6)表面等离子体共振测定HSA-Ω及Ω-HSA与suPAR结合的动力学参数
通过表面等离子体共振实验测定HSA-Ω与Ω-HSA直接结合suPAR的动力学参数,比较二者结合suPAR的能力。将suPAR固定在CM5传感芯片上(缓冲液为10 mM sodiumacetate pH 5.0)，用HBS缓冲溶液(20 mM HEPES PH 7.4, 150 mM NaCl) 三倍梯度稀释的融合蛋白流过芯片表面(25℃下以30 μL/min的流速注入120 s)与suPAR相互作用，使用BIACORE评价软件，采用1:1动力学拟合，计算动力学参数 (kon和koff)，KD值通过koff/kon计算得到。结果表明，HSA-Ω与suPAR结合的KD值为690 nM，而 Ω-HSA 则完全没有结合suPAR的能力 (KD值超过500 μM)。
表1 表面等离子体共振测定HSA-Ω与suPAR结合的动力学参数
实施例二：HSA与多肽IG2的融合
为了验证该融合方法是否对其他多肽有效，我们将另一多肽IG2与HSA进行融合。该多肽氨基酸序列为CPAYSRYIGC，是鼠源uPA (murine uPA, muPA) 的特异性抑制剂，对muPA 水解其发色底物S-2444的抑制常数为20 nM。本实施例中，将IG2分别融合在HSA的A363与A364之间以及加入连接序列GGGGSGGGGSGGGGS后融合在HSA的N端，得到融合蛋白HSA-IG2与IG2-HSA。
(1)HSA-IG2和IG2-HSA表达载体的构建，蛋白的表达与纯化及二级结构的表征
按照实施例一相同的方法，构建HSA-IG2和IG2-HSA的表达载体。蛋白的表达与纯化如实施例一所述，SDS-PAGE分析结果如图6A，HSA-IG2与IG2-HSA 在酵母系统中表达产率与纯度与野生型HSA一致; HSA-IG2与HSA的圆二色谱结果如图6B, 二者的α-螺旋含量基本一致，表明IG2的插入不影响HSA的二级结构。
(2)HSA-IG2 与 IG2-HSA 对muPA抑制能力的测定
muPA水解其发色底物S-2444后，在405 nm 波长处产生吸收。基于此，测定在不同浓度融合蛋白下，muPA水解S-2444的反应速度，反应融合蛋白对muPA的抑制能力。测定步骤如下：在384孔透明板中分别加入muPA（终浓度为5 nM）和三倍梯度稀释的HSA-IG2 或 IG2-HSA (150 µM - 0.01 nM)，室温孵育15 min之后，加入底物S-2444，测定405 nm 波长处的吸收值，监测反应速率(检测总时长为45 min，间隔45 s)。反应速率对LogC进行非线性拟合，确定 HSA-IG2与 IG2-HSA抑制muPA水解S-2444的IC50值。如图6C所示，HSA-IG2与游离的IG2多肽对muPA的抑制能力相当，IC50值分别为32 nM与24 nM，明显强于 IG2-HSA(IC50值为19000 nM)。
上述实例表明，将生物活性肽融合在HSA内部特定的转弯上是一种通用的融合方法，该方法不仅可以保持HSA的结构与功能，发挥HSA作为药物载体的优势，相比于将多肽融合在HSA末端，该方法还能够维持多肽的高生物活性，因此可实现在维持多肽高活性的同时，改善其药代动力学性质，赋予其更好的成药性。
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<212> PRT
<213> 人工序列
<400> 13
Cys Pro Ala Tyr Ser Arg Tyr Ile Gly Cys
1 5 10
<210> 14
<211> 15
<212> PRT
<213> 人工序列
<400> 14
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
Claims (1)
1.一种在人血清白蛋白内部融合多肽的方法，其特征在于：将具有生物活性的多肽融合在人血清白蛋白HSA结构域Ⅱ的两条α螺旋链之间转弯C360-C369位点上；
所述转弯C360-C369位点，附近存在两对二硫键C360-C369和C361-C316；且该转弯位于HSA表面并向外凸出；该转弯远离HSA与Fc受体的结合位点；
所述具有生物活性的多肽包括为氨基酸序列为CVSNKYFSNIHWC的多肽；
所述方法包括以下步骤：
（1）内部融合位点选择：内部融合位点选择位于HSA结构域Ⅱ的两条α螺旋链之间转弯C360-C369上；
（2）内部融合蛋白的构建和表达：多肽编码基因融合到HSA转弯C360-C369的基因位置上，并在真核表达系统中进行高效表达，得到融合蛋白HSA-Ω，氨基酸序列如SEQ ID NO.1所示；
所述多肽编码基因融合的方法为LIC方法；
所述真核表达系统为酵母表达系统。
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| CN104800855A (zh) * | 2014-01-24 | 2015-07-29 | 中国科学院福建物质结构研究所 | 用于光学成像和光动力治疗的肿瘤靶向融合蛋白药物载体 |
| CN106796222A (zh) * | 2014-06-20 | 2017-05-31 | 乌尔里希·洛斯 | 针对tsh受体的自身抗体的检测 |
| CN112180103A (zh) * | 2020-10-23 | 2021-01-05 | 福州大学 | 一种用于临床检测新冠肺炎患者血浆中活性尿激酶受体的试剂盒 |
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| CN104800855A (zh) * | 2014-01-24 | 2015-07-29 | 中国科学院福建物质结构研究所 | 用于光学成像和光动力治疗的肿瘤靶向融合蛋白药物载体 |
| CN106796222A (zh) * | 2014-06-20 | 2017-05-31 | 乌尔里希·洛斯 | 针对tsh受体的自身抗体的检测 |
| CN112180103A (zh) * | 2020-10-23 | 2021-01-05 | 福州大学 | 一种用于临床检测新冠肺炎患者血浆中活性尿激酶受体的试剂盒 |
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