CN114539383B - 一种人源膜联蛋白人源膜联蛋白a5突变体二聚体的晶体结构的制备方法和应用 - Google Patents
一种人源膜联蛋白人源膜联蛋白a5突变体二聚体的晶体结构的制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种人源膜联蛋白A5突变体二聚体的晶体结构的制备方法，包括如下步骤：（1）将人源膜联蛋白A5的基因序列通过EcoRⅠ和Xhol Ⅰ酶切位点连接至pET‑28a(+)质粒上，将表达载体转入宿主大肠杆菌BL21(DE3)中表达，通过亲和层析纯化，获得高纯度蛋白；（2）将蛋白浓缩为10 mg/mL的蛋白水溶液，将蛋白溶液与池液混合，采取坐滴法对结晶条件进行高通量筛选；对结晶条件进行正交优化，制得人源膜联蛋白A5突变体二聚体的晶体结构。本发明所提供的突变体蛋白的结构信息可以用于更高效地开展制备检测试剂、诊断试剂盒制备、药物靶点筛选和治疗效果更好的变体蛋白研发，满足生物制药应用的要求。
Description
技术领域
本发明涉及医药工程技术领域，具体涉及一种人源膜联蛋白A5突变体二聚体的晶体结构的制备方法和应用。
背景技术
研究表明，膜联蛋白A5（Annexin A5）简称ANXA5，是一种320个氨基酸组成的单链蛋白，研究早期其又被称为胎盘抗凝蛋白 I、凝血酶抑制剂 V、内皮素 II、钙结合蛋白 I和脂皮质素 V。早在 1985 年，研究报道将其描述为血管抗凝剂，之后它又成为第一个被表征三维结构的膜联蛋白，表明对其的结构解析具有十分重要的研究价值。
研究表明，ANXA5蛋白结构重复序列 I 至 IV 组成，其蛋白序列 226 位上的 ASP以Ca2+和 pH 依赖的构象方式参与调控分子开关。ANXA5与 Ca2+结合时的构象，发现在ANXA5 重复序列 III 第 187 位上暴露的 Trp 与磷脂结合，此外，ANXA5 内部的域间形成了由 I/IV 和 II/III 组成的交互作用方式。其中，I和 IV 之间的相互作用是由 NH2 末端以非共价方式介导产生，而重复序列 II 和III 通过短螺旋转动方式共价连接。
研究表明， ANXA5 是一种单体结构，但与磷脂膜结合时，三个 ANXA5 单体会自发形成三聚体，而形成的三聚体可以通过三聚体-三聚体相互作用的方式在暴露有磷脂酞丝氨酸（PS）的表面组装。在涉及到抗炎、纤溶和抗血栓过程中，细胞内的 ANXA5 会在二维晶格中自组装，进而改变细胞膜的曲率以及细胞形状，最终促进细胞膜的修复。
研究表明，细胞内的 ANXA5 在质膜上表现出钙通道活性，它能与血小板中的肌动蛋白相互作用，是凝血过程的关键调节因子。另外，在血液凝固过程中需要血小板的激活，以形成一个以PS为主的磷脂膜催化表面，使凝血因子X、凝血酶原等凝血固子活化。由于膜联蛋白具有较强的钙依赖性磷脂结合活性，因而能够竞争性抑制凝血因子和血小板膜磷脂的结合，从而抑制了凝血因子X等的活化，起到抗凝血的作用。
综上所述，Annexins是一类钙离子依懒型的磷脂结合蛋白，能够调控抗凝血、细胞凋亡、信号转导等过程，并参与多种疾病的发生发展。ANXA5是膜联蛋白家族分布最多和最广泛的成员，最早被称为抗凝蛋白，主要起抗凝血的功能，通常作为抗凝剂和抗血栓形成的药。近几年，对于寻找与PS结合能力更强、抗血栓能力更强和毒性更小的ANXA5突变体成为研究热点。本发明提供了一个构建此类突变体的新思路，即通过在ANXA5的C末端添加一个半胱氨酸，能够通过形成分子间的二硫桥的构建提高分子间的聚集作用，在竞争性抑制凝血因子和血小板膜磷脂的结合中发挥更强的作用。
目前，X-射线单晶衍射方法是通过蛋白质单晶获得其三维结构的最重要的研究方法之一。对于X-射线单晶衍射方法，蛋白质单晶必须达到足够大的尺寸和完善程度。然而，由于溶液中蛋白质分子之间的相互作用位点较少，相互作用力比较弱，蛋白质晶体中往往含有很大比重的水，因此，内部结构比较规整的蛋白质晶体仍旧很难获得。获得高质量的蛋白质晶体仍旧是蛋白质结构解析的瓶颈问题。
发明内容
本发明提供了一种人源膜联蛋白A5突变体二聚体的晶体结构的制备方法和应用。
为了解决现有技术的问题，本发明提供了如下技术方案：本发明的一种所述的人源膜联蛋白A5突变体二聚体的晶体结构的制备方法，包括如下步骤：（1）将人源膜联蛋白的基因序列通过EcoRⅠ和Xhol Ⅰ酶切位点连接至pET-28a(+)质粒上，将表达载体转入宿主大肠杆菌BL21(DE3)中表达，通过亲和层析纯化，获得高纯度蛋白；
（2）将蛋白浓缩为10 mg/mL的蛋白水溶液，将蛋白溶液与池液混合，采取坐滴法对结晶条件进行高通量筛选；对结晶条件进行正交优化，制得人源膜联蛋白A5突变体二聚体的晶体结构。
进一步地，在步骤（1）中，将人源膜联蛋白A5突变体二聚体基因序列与pET-28a(+)质粒相连接，将表达载体转入宿主菌BL 21(DE 3)中表达，可获得可溶表达产物；培养条件是：采用LB培养基培养，按照2%比例扩大培养，培养到一定时间加入IPTG诱导，诱导时间为20 h；离心收集菌体超声破碎，离心收集上清液，用亲和层析柱进行蛋白粗酶液的纯化，最后用10 KD横纵切向流超滤膜获得浓缩后的高浓度蛋白溶液；利用Hiload 16/60 Superdex200分子筛进一步纯化蛋白，实验流程为：先用1.2倍柱体积的Buffer（10 mM Tris，100 mMNaCl，pH 8.5）平衡分子筛，流速1 mL/min；将5 mL 离心后的蛋白加载到分子筛，收集洗脱的蛋白，并用SDS-PAGE鉴定。
在步骤（2）中，将分子筛纯化后的蛋白浓缩到 10 mg/mL，用坐滴法对初始结晶条件进新了高通量筛选, 共筛选出了10个结晶kit；2天后，在含有0.2 µl 蛋白和0.2 µl 池液的结晶液滴中长出了晶体；对培养条件进行优化，优化后，含有2 µl 蛋白溶液和1 µl 池液的结晶液滴中长出的晶体符合要求。
进一步地，在步骤（2）中，所述的池液含有0.2 M氯化钠，0.1 M 4-羟乙基哌嗪乙磺酸的pH为 6.5-7.5和16%-26%聚乙二醇单甲醚3000。
进一步地，在步骤（2）中，所述的池液含有0.2 M氯化钠，0.1 M 4-羟乙基哌嗪乙磺酸pH 7.2和22%聚乙二醇单甲醚3000。
本发明所述的一种所述的人源膜联蛋白A5突变体二聚体的晶体结构的制备方法所制备和解析的人源膜联蛋白A5突变体二聚体的晶体结构在制备检测试剂、诊断试剂盒以及药物中的应用。
本发明的一种人源膜联蛋白突变体二聚体的晶体结构的制备方法，所述的人源膜联蛋白A5突变体二聚体的晶体结构的氨基酸序列如SEQ ID NO.1所示。
所述的人源膜联蛋白A5突变体二聚体的晶体结构的一个晶格由4个蛋白分子组成，分别为：chain A、chain B、chain C、chain D。
有益效果：本发明可以用于设计和/或修改膜联蛋白A5以提高其抗凝血、抗炎症、识别凋亡细胞等作用，提高稳定性等方面，满足生物制药应用的要求。
与现有技术相比，本发明具有如下优点：
（1）本发明在分子水平的基础上解析了人源膜联蛋白A5突变体二聚体的晶体结构。晶体结构及其衍生出的信息适用于设计和鉴定新药物，适用于设计具有高稳定性和药效的突变体蛋白。本发明可用于诸如，改善蛋白稳定性、提高药物疗效、降低药物毒性、构建ANXA5突变体库、医药领域筛选抑制剂等方面。
（2）本发明提供了解析膜联蛋白晶体结构的方法，通过该方法成功获得人源膜联蛋白ANXA5突变体二聚体的晶体结构。本发明公布的人源膜联蛋白A5突变体二聚体晶体结构信息可用于解释基于构效关系的膜联蛋白抗凝血、抗炎症、识别凋亡细胞等活性的机制。本发明提供的ANXA5突变体结构完整，保留氨基酸信息全面，为ANXA5的研究提供了更加详细的结构信息。
附图说明
图1为本发明的人源膜联蛋白A5突变体二聚体经过分子筛进一步纯化之后的SDS-PAGE电泳结果。
图2为本发明的人源膜联蛋白A5突变体二聚体高通量筛选的片状晶体示意图。
图3为本发明的人源膜联蛋白A5突变体二聚体优化后的块状晶体示意图。
具体实施方式
下面通过实施例对本发明做进一步说明，但本发明不受实施例的限制。
实施例1
本发明的一种人源膜联蛋白A5突变体二聚体的晶体结构，采用技术方案如下：本发明的一种人源膜联蛋白A5突变体二聚体的晶体结构，所述的人源膜联蛋白A5突变体二聚体的晶体结构的氨基酸序列如SEQ ID NO.1所示。
所述的人源膜联蛋白A5突变体二聚体的晶体结构的一个晶格由4个蛋白分子组成，分别为：chain A、chain B、chain C、chain D。
本发明的一种人源膜联蛋白A5突变体二聚体的晶体结构的制备方法，包括如下步骤：（1）将人源膜联蛋白A5突变体的基因序列通过EcoRⅠ和Xhol Ⅰ酶切位点连接至pET-28a(+)质粒上，将表达载体转入宿主大肠杆菌BL21(DE3)中表达，通过亲和层析纯化，获得高纯度蛋白；
（2）将蛋白浓缩为10 mg/mL的蛋白水溶液，将蛋白溶液与池液混合，采取坐滴法对结晶条件进行高通量筛选；对结晶条件进行正交优化，制得人源膜联蛋白A5突变体二聚体的晶体结构。所述的池液含有0.2 M氯化钠，0.1 M 4-羟乙基哌嗪乙磺酸的pH为 6.5和16%聚乙二醇单甲醚3000。
本发明的一种人源膜联蛋白A5突变体二聚体的晶体结构的制备方法，包括如下步骤：在步骤（1）中，将人源膜联蛋白A5突变体基因序列与pET-28a(+)质粒相连接，将表达载体转入宿主菌BL 21(DE 3)中表达，可获得可溶表达产物；培养条件是：采用LB培养基培养，按照2%比例扩大培养，培养到一定时间加入IPTG诱导，诱导时间为20 h；离心收集菌体超声破碎，离心收集上清液，用亲和层析柱进行蛋白粗酶液的纯化，最后用10 KD横纵切向流超滤膜获得浓缩后的高浓度蛋白溶液；利用Hiload 16/60 Superdex 200分子筛进一步纯化蛋白，实验流程为：先用1.2倍柱体积的Buffer（10 mM Tris，100 mM NaCl，pH 8.5）平衡分子筛，流速1 mL/min；将5 mL 离心后的蛋白加载到分子筛，收集洗脱的蛋白，并用SDS-PAGE鉴定。
在步骤（2）中，将分子筛纯化后的蛋白浓缩到 10 mg/mL，用坐滴法对初始结晶条件进新了高通量筛选, 共筛选出了10个结晶kit；2天后，在含有0.2 µl 蛋白和0.2 µl 池液的结晶液滴中长出了晶体；对培养条件进行优化，优化后，含有2 µl 蛋白溶液和1 µl 池液的结晶液滴中长出的晶体符合要求。所述的池液含有0.2 M氯化钠，0.1 M 4-羟乙基哌嗪乙磺酸pH 7.2和22%聚乙二醇单甲醚3000。
本发明所述的人源膜联蛋白人源膜联蛋白A5突变体二聚体的晶体结构在制备检测试剂、诊断试剂盒以及药物中的应用。
实施例2
实施例2与实施例1的区别在于：
所述的池液含有0.2 M氯化钠，0.1 M 4-羟乙基哌嗪乙磺酸的pH为 7.5和26%聚乙二醇单甲醚3000。
试验例1
人源膜联蛋白A5突变体二聚体的表达与纯化
人工合成编码人源膜联蛋白A5突变体的 DNA序列。用双酶切位点（EcoRⅠ和XholⅠ）连入 pET-28 a（+）载体中。将重组质粒转化大肠杆菌 BL 21(DE3) 感受态细胞，将所得到的转化子克隆挑取转接入适量 LB 培养基（加入卡那霉素至终浓度为50 μg/mL）中，37℃培养至 600 nm 吸光度为0.8，加入诱导剂异丙基-β-D-硫代吡喃半乳糖苷（IPTG）至终浓度为 0.5 mM，20℃诱导20 h。以4200 r/min离心30 min 收取菌体后，用缓冲液：20 mM Tris-HCl（三羟甲基氨基甲烷-盐酸），pH 8.0 以菌体：缓冲液=1（g）：10（mL）比例加入缓冲液使菌体重悬。菌体重悬，高压破碎后，以12000 r/min低温离心40 min去除沉淀和其他颗粒状杂质。将离心后上清液与Ni-NTA亲和介质结合后，用含有500 mM氯化钠、100 mM磷酸盐缓冲液、20 mM咪唑的缓冲液冲洗介质，去除杂蛋白。最终用含有500 mM氯化钠、100 mM磷酸盐缓冲液、500 mM咪唑的洗脱液将目的蛋白从亲和介质上洗脱下来，收集各个不同浓度洗脱液的峰尖蛋白，利用SDS-PAGE电泳检测。以10 KD横纵切向流超滤膜将洗脱液浓缩并更换至20mM Tris-HCl，pH 8.5的缓冲液里储存，用液氮速冻后放置-80℃冰箱保存。
试验例2
人源膜联蛋白A5突变体二聚体晶体的筛选与优化
缓冲液: 10 mM Tris-HCl、100 mM NaCl pH 8.5；分子筛：Hiload 16/60Superdex 200
实验流程：先用1.2倍柱体积的缓冲液平衡分子筛，流速为1 mL/min；将5ml 离心后的蛋白加载到分子筛，收集洗脱的蛋白，并用SDS-PAGE电泳鉴定 (结果如图1所示)。
将经过分子排阻层析纯化后的蛋白浓缩到 10 mg/mL，用坐滴法对初始结晶条件进新了高通量筛选，共筛选了10个结晶kit。2天后，于16摄氏度，在含有0.2 µl 蛋白和0.2µl 池液(0.2 M 氯化钠， 0.1 M HEPES pH 6.5，26 % （w/v） Polyethylene glycolmonomethyl ether 3000) 的结晶液滴中长出了晶体。 优化后，含有2 µL蛋白和1 µl池液(0.2 M 氯化钠，0.1 M HEPES pH 7.2，26% (w/v) Polyethylene glycol monomethylether 3000)的结晶液滴中长出了形状较好的晶体 (结果如图2、图3所示)。
试验例3
人源膜联蛋白A5突变体二聚体晶体结构的解析
从母液中可以获得晶体，利用液态氮气流快速冷冻，在100 K下利用X射线源获得的晶体数据，具体过程如下：
首先使用的尼龙晶体环（如Hampton Research公司的），从晶体浸泡液中获取适宜X射线衍射的晶体，并迅速使用冷却系统（如Oxford Cryosystem公司的）产生的低温氮气流中冷冻至零下150-180℃；使X射线通过晶体，利用旋进法收集X射线衍射数据。收集到晶体的X射线衍射数据后，按照下述步骤进行相应的数据处理：首先使用HKL2000等软件对上一步骤中收集得到的衍射数据进行处理，获得完整的数据文件；其次，使用CCP4程序包中的Phaser、Molrep等软件，利用分子置换（MR，Molecular Replacement）方法，以已知的膜联蛋白的结构（1ANX）为搜索模型，得到人源膜联蛋白A5突变体二聚体的初始结构，再继续使用PHENIX软件对人源膜联蛋白A5突变体二聚体的初始结构进行结构优化。
人源膜联蛋白A5突变体二聚体晶体一个晶格含有4个蛋白分子，分别为：chain A、chain B、chain C、chain D。
试验例4
人源膜联蛋白A5突变体二聚体的晶体空间结构特征
该人源膜联蛋白A5突变体二聚体晶体每个晶格有4个蛋白分子，分别为chain A、chain B、chain C、chain D。每个单分子由322个氨基酸组成，含有4个repeat重复单元。
以上所述，对本发明通过说明及具体实施方式进行了阐述，在本发明的基础上，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，根据本发明的技术方案及其发明构思加以等同替换或改变，都应涵盖在本发明的保护范围之内。
序列表
<110> 南京大学，南京吉芮康生物科技研究院有限公司
<120> 一种人源膜联蛋白A5突变体二聚体的晶体结构的制备方法及其应用
<130> 2022
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 322
<212> PRT
<213> 人工序列(人源膜联蛋白A5突变体二聚体的晶体结构的氨基酸序列)
<400> 1
Met Gly Ala Gln Val Leu Arg Gly Thr Val Thr Asp Phe Pro Gly Phe
1 5 10 15
Asp Glu Arg Ala Asp Ala Glu Thr Leu Arg Lys Ala Met Lys Gly Leu
20 25 30
Gly Thr Asp Glu Glu Ser Ile Leu Thr Leu Leu Thr Ser Arg Ser Asn
35 40 45
Ala Gln Arg Gln Glu Ile Ser Ala Ala Phe Lys Thr Leu Phe Gly Arg
50 55 60
Asp Leu Leu Asp Asp Leu Lys Ser Glu Leu Thr Gly Lys Phe Glu Lys
65 70 75 80
Leu Ile Val Ala Leu Met Lys Pro Ser Arg Leu Tyr Asp Ala Tyr Glu
85 90 95
Leu Lys His Ala Leu Lys Gly Ala Gly Thr Asn Glu Lys Val Leu Thr
100 105 110
Glu Ile Ile Ala Ser Arg Thr Pro Glu Glu Leu Arg Ala Ile Lys Gln
115 120 125
Val Tyr Glu Glu Glu Tyr Gly Ser Ser Leu Glu Asp Asp Val Val Gly
130 135 140
Asp Thr Ser Gly Tyr Tyr Gln Arg Met Leu Val Val Leu Leu Gln Ala
145 150 155 160
Asn Arg Asp Pro Asp Ala Gly Ile Asp Glu Ala Gln Val Glu Gln Asp
165 170 175
Ala Gln Ala Leu Phe Gln Ala Gly Glu Leu Lys Trp Gly Thr Asp Glu
180 185 190
Glu Lys Phe Ile Thr Ile Phe Gly Thr Arg Ser Val Ser His Leu Arg
195 200 205
Lys Val Phe Asp Lys Tyr Met Thr Ile Ser Gly Phe Gln Ile Glu Glu
210 215 220
Thr Ile Asp Arg Glu Thr Ser Gly Asn Leu Glu Gln Leu Leu Leu Ala
225 230 235 240
Val Val Lys Ser Ile Arg Ser Ile Pro Ala Tyr Leu Ala Glu Thr Leu
245 250 255
Tyr Tyr Ala Met Lys Gly Ala Gly Thr Asp Asp His Thr Leu Ile Arg
260 265 270
Val Met Val Ser Arg Ser Glu Ile Asp Leu Phe Asn Ile Arg Lys Glu
275 280 285
Phe Arg Lys Asn Phe Ala Thr Ser Leu Tyr Ser Met Ile Lys Gly Asp
290 295 300
Thr Ser Gly Asp Tyr Lys Lys Ala Leu Leu Leu Leu Cys Gly Glu Asp
305 310 315 320
Asp Cys
Claims (2)
1.一种人源膜联蛋白A5突变体二聚体蛋白晶体的制备方法，其特征在于包括如下步骤：（1）将人源膜联蛋白A5突变体的编码基因序列通过EcoRⅠ和Xhol Ⅰ酶切位点连接至pET-28a(+)质粒上，将表达载体转入宿主大肠杆菌BL21(DE3)中表达，通过亲和层析纯化，获得高纯度蛋白，所述人源膜联蛋白A5突变体的氨基酸序列如SEQ ID NO.1所示；
（2）将蛋白浓缩为10 mg/mL的蛋白水溶液，将蛋白溶液与池液混合，采取坐滴法对结晶条件进行高通量筛选；对结晶条件进行正交优化，优化后的结晶液滴含有2ul蛋白溶液和1ul池液；所述的池液含有0.2M氯化钠，0.1M 4-羟乙基哌嗪乙磺酸pH 7.2和26%聚乙二醇单甲醚3000，制得人源膜联蛋白A5突变体二聚体蛋白晶体。
2.根据权利要求1所述的人源膜联蛋白A5突变体二聚体蛋白晶体的制备方法，其特征在于：在步骤（1）中，将人源膜联蛋白A5突变体的编码基因序列与pET-28a(+)质粒相连接，将表达载体转入宿主菌BL21(DE3)中表达，可获得可溶表达产物；培养条件是：采用LB培养基培养，按照2%比例扩大培养，培养到一定时间加入IPTG诱导，诱导时间为20 h；离心收集菌体超声破碎，离心收集上清液，用亲和层析柱进行蛋白粗酶液的纯化，最后用10 KD横纵切向流超滤膜获得浓缩后的高浓度蛋白溶液；利用Hiload 16/60 Superdex 200分子筛进一步纯化蛋白，实验流程为：先用1.2倍柱体积的Buffer平衡分子筛，流速1 mL/min；将5 mL离心后的蛋白加载到分子筛，收集洗脱的蛋白，并用SDS-PAGE鉴定。
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