药学学报  2014, Vol. 49 Issue (8): 1200-1207   PDF    
LEDGF/p75蛋白的可溶性表达、纯化及功能研究
张大为1, 何红秋2, 郭顺星1     
1. 中国医学科学院、北京协和医学院药用植物研究所, 北京 100193;
2. 重庆市科学技术学院重庆生物医药与器械研究中心, 重庆 401123
摘要:HIV-1整合酶(integrase,IN) 是病毒复制过程中的一个关键酶,其与宿主晶状体上皮源性生长因子p75(lens epithelium-derived growth factor p75,LEDGF/p75) 的蛋白-蛋白相互作用是筛选抗病毒药物的一个重要靶点。为开展以IN-LEDGF/p75相互作用为靶点的抑制剂研究,本研究构建了LEDGF/p75蛋白重组质粒,在原核细胞中进行了可溶性表达和功能研究。根据大肠杆菌密码子偏爱性,全合成高利用率密码子的LEDGF/p75基因序列,并克隆到表达载体pGEX-4T-1中构建重组质粒。在大肠杆菌中优化表达LEDGF/p75蛋白,经SDS-PAGE鉴定和亲和色谱纯化蛋白,采用酶联免疫吸附实验方法(ELISA) 测定了LEDGF/p75蛋白的生物学活性。结果显示,构建的重组质粒获得高效稳定的可溶性表达,ELISA证实体外表达的LEDGF/p75蛋白能够与HIV-1 IN相互作用,并促进IN链转移反应的完成。本研究为建立以LEDGF/p75-IN相互作用为靶点的抗HIV药物筛选平台打下了基础。
关键词LEDGF/p75     基因表达     HIV-1整合酶     ELISA    
Prokaryotic soluble expression, purification and function study of LEDGF/p75 protein
ZHANG Da-wei1, HE Hong-qiu2, GUO Shun-xing1     
1. Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China;
2. Chongqing Center for Biomedicines and Medical Equipment, Chongqing Academy of Science and Technology, Chongqing 401123, China
Abstract: HIV-1 integrase (IN) is a key enzyme for the viral replication. The protein-protein interaction (PPI) between HIV-1 IN and a cellular cofactor lens epithelium-derived growth factor (LEDGF/p75) is a validated target for anti-HIV drug discovery. In order to build the platform for screening inhibitor against PPI between IN and LEDGF/p75, the vector containing the LEDGF/p75 protein cDNA was constructed and expressed in Escherichia coli and the function of the LEDGF/p75 protein was assayed. The LGDGF/p75 encoding gene optimized according to the preference codon usage of E. coli, was synthesized and cloned into the expression vector pGEX-4T-1 to form a recombined plasmid, then trans-formed into host cell E. coli BL21 (DE3). The recombined clones were identified and confirmed by BamH I/Sal I digestion and sequencing, the successfully recombined plasmid in the host cell was induced by IPTG and the condition of the expression was optimized. The expressed protein was purified by the Ni2++ affinity chromatography column and SDS-PAGE was used to analyze the molecular weight and specificity. In addition, ELISA assay was used to analyze the function of the recombinant protein. The recombinant LGDGF/p75 was soluble, and expressed highly and stably in E. coli. The protein was proved to en-hance HIV-1 IN strand transfer activity in vitro by ELISA. It will be helpful to build the platform of screening inhibitors against PPI between IN and LEDGF/p75.
Key words: LEDGF/p75     gene expression     HIV-1 integrase     ELISA    

1998~2003年,来自转录调控、细胞存活、自体免疫以及病毒学4个不同领域的科学家独立地纯化到一种分子质量约为75 kDa的蛋白,该蛋白能够促进转录活性,是一种转录协同激活因子 (p75)[1]。后来,研究者又在老鼠的晶状体上皮细胞中分离到这种蛋白,该蛋白因此被命名为晶状体上皮源性生长因子p75 (lens epithelium-derived growth factor p75)[2]。LEDGF/p75由530个氨基酸残基组成,根据其功能可以划分为2个区域,即N端DNA/染色体结合区和C端整合酶结合区 (integrase-binding domain,IBD)[3]。实际上,LEDGF/p75既非生长因子,也无组织特异性表达,广泛存在于人体各种细胞中,对细胞存活与凋亡起重要调节作用[4]

2003年,Maertens等[5]通过免疫共沉淀的方法首先发现了LEDGF/p75与HIV-1整合酶 (integrase,IN) 的蛋白相互作用 (protein-protein interaction,PPI),并证明这种PPI对于IN进入细胞核以及染色体定位是必须的。随后,LEDGF/p75蛋白编码基因的突变、RNAi、敲除实验以及IBD结构域的过表达实验证明,LEDGF/p75与IN之间的PPI是有效的抑制剂筛选靶点[6,7,8,9,10,11,12,13],并且大量具有抑制活性的化合物被筛选出来,如D77、BI-1001、CHIBA3053、CX0516、CX05045、CX14442[14,15,16,17,18]等。其中,化合物CX14442在细胞水平的选择性指数达到1 391,显示了良好的应用前景[14]。此外,随着第一代IN抑制剂临床耐药性的出现[19,20],开发具有新作用机制的IN抑制剂显得尤为迫切。因此,以LEDGF/p75-IN相互作用为靶点的抗HIV药物开发具有重要意义。

LEDGF/p75是一种人源蛋白,为了获得可溶表达且具有活性的重组LEDGF/p75蛋白,开展以LEDGF/ p75-IN相互作用为靶点的抑制剂筛选研究,本文通过全合成根据大肠杆菌密码子偏爱性优化后的LEDGF/ p75编码基因,构建重组表达质粒,在大肠杆菌中融合表达,并测定了LEDGF/p75蛋白的生物活性。

材料与方法 材料与试剂

大肠杆菌 (Escherichia coli) 菌株BL21、BL21 (DE3)、BL21 (DE3) pLysS购自北京全式金生物技术有限公司。质粒pGEX-4T-1、C-terminal His-taged HIV-1 IN (IN-His) 由本实验室保存。GST琼脂糖凝胶FF色谱柱购自北京韦氏博慧色谱科技有限公司。各种限制性内切酶、DNA连接酶、Taq酶,DNA分子量标准、蛋白质分子量标准均购自宝生物工程 (大连) 有限公司。Dynabeads M-280 streptavidin magnetic particle (链霉亲和素磁珠) 和Dynal MPC-9 concentrator (板式磁珠收集器) 购自美国Dynal Biotech公司。Monoclonal anti-dioxin alkaline phosphatase conjugated antibody (碱性磷酸酶标记地高辛抗体) 和4-nitrophenyl phosphate salt (p-NPP) 购自美国Sigma公司。96孔透明微孔板购自丹麦NUNC公司。

链转移反应底物的制备

寡核苷酸链STD1 5'- biotin-ACCCTTTTAGTCAGTGTGGAA-3',STD2 5'- ACTGCTAGATTTTCCACACTGACTAAAAG-3',STT1 5'-TGACCAAGGGCTAATTCACT-digoxin-3',STT2 5'- AGTGAATTAGCCCTTGGTCA-3'由上海生工生物工程技术服务有限公司合成和修饰标记。STD1和STD2等摩尔量混合退火形成链转移反应供体DNA (STD-DNA),STT1和STT2等摩尔量混合退火形成链转移反应靶DNA (STT-DNA)。制备条件是95 ℃变性3 min,缓慢冷却退火至室温[21]

LEDGF/p75基因合成

参照野生型LEDGF/p75基因序列 (GenBank注册号: AF199339),结合大肠杆菌的密码子偏爱性进行优化,将其中的稀有密码子突变为偏爱密码子。合成基因,同时在5'-端添加BamH I酶切位点,在3'-端添加Sal I序列。将合成的LEDGF/ p75克隆至pUCm-T载体,转化大肠杆菌DH5α感受态细胞。挑取克隆进行DNA序列测定(苏州金维智),测序正确的重组质粒命名为pUCm-LEDGF/p75。

BamH I和Sal I双酶切重组质粒pUCm-IN获取LEDGF/p75基因,连接到同样双酶切的pGEX-4T- 1表达载体,转化DH5α感受态细胞,筛选阳性克隆并提取质粒测序鉴定,正确构建的含LEDGF/p75基因的重组表达质粒命名为pGEX-4T-p75。

细菌培养

将测序正确的pGEX-4T-p75重组质粒转化大肠杆菌宿主菌,涂布于含100 µg∙mL-1氨苄西林的LB固体培养基上,37 ℃培养过夜,挑取单个克隆接种于3 mL含100 µg∙mL-1氨苄青霉素的LB 液体培养液中,37 ℃、180 r∙min-1振荡培养12~16 h,用作种子液。种子液按照1∶100的比例分别接种于含100 µg∙mL-1氨苄青霉素的液体LB中,37 ℃振荡培养至对数中期,以备诱导表达蛋白。

LEDGF/p75蛋白诱导表达条件优化

分别考察了宿主菌株、诱导温度、诱导时间对蛋白表达的影 响。宿主菌选取E. Coli BL21、BL21 (DE3)、BL21 (DE3) pLysS 3个菌株进行考察。诱导温度选择37 ℃和30 ℃两个温度进行考察。诱导时间选择1、3、4和5 h 4个时间点进行考察。

目的蛋白的可溶性考察

将转化有pGEX-4T- p75的BL21 (DE3) 种子液,37 ℃振荡培养至对数中期,加入诱导剂IPTG至终浓度为0.5 mmol∙L-1,30 ℃诱导表达3 h。离心收集菌体。超声破碎菌体,离心,收集上清液。沉淀用PBS重悬,SDS-PAGE分析蛋白的可溶性。

p75蛋白的纯化

将诱导表达的培养物离心,收集菌体,加入4 ℃预冷的裂解液 (50 mmol∙L-1 Tris- HCl,pH 7.2,500 mmol∙L-1 NaCl,1 mmol∙L-1 DTT,1 mmol∙L-1 EDTA,1 mmol∙L-1 PMSF) 重悬菌体,冻融3次 (-80 ℃/4 ℃),冰浴超声破菌,12 000 r·min-1离心,收集上清液,上清液以0.45 μm滤膜过滤。利用 GST琼脂糖凝胶FF亲和色谱柱纯化LEDGF/p75融合蛋白。吸取过滤的上清液过buffer A (50 mmol∙L-1 Tris-HCl,pH 8.0,500 mmol∙L-1 NaCl,1 mmol∙L-1 DTT) 平衡后的亲和柱,再以缓冲液A洗去结合的非特异性蛋白,以缓冲液B (50 mmol∙L-1 Tris-HCl,pH 8.0,500 mmol∙L-1 NaCl,1 mmol∙L-1 DTT,20 mmol∙L-1还原型谷胱甘肽) 洗脱目的蛋白,收集洗脱峰对应的蛋白。超滤,置换缓冲液为缓冲液C (25 mmol∙L-1 HEPES pH 7.3,500 mmol∙L-1 NaCl,1 mmol∙L-1 DTT,10% Glycerin)。SDS-PAGE分析表达纯化效果,BCA法测定蛋白浓度[22]

LEDGF/p75蛋白对IN链转移反应活性的影响

活性测定采用He等[21]报道的基于磁珠的ELISA,反应体系为50 μL。将200 nmol∙L-1 HIV-1 IN蛋白、200 nmol∙L-1 p75/LEDGF蛋白和30 nmol∙L-1的STD- DNA在Reaction buffer (25 mmol∙L-1 PIPES,pH 7.0,10 mmol∙L-1 β-mercaptoethanol,0.1 g∙L-1 BSA,5% Glycerin) 中混匀,室温孵育15 min。各孔中加入5 μL 3% 的DMSO,混匀,37 ℃反应1 h。加入300 nmol∙L-1 的STT-DNA,混匀,37 ℃反应2 h。加入1.5 μL链霉亲和素磁珠和51.5 μL结合缓冲液 [10 mmol∙L-1 Tris- HCl,pH 7.6,2 mol∙L-1 NaCl,20 mmol∙L-1 EDTA,0.1% (w/v) 吐温20],室温孵育15 min,将微孔板置于磁珠收集器上90 s,收集磁珠,弃上清液。用100 μL TBST (20 mmol∙L-1 Tris,pH 7.4,137 mmol∙L-1 NaCl,0.1% 吐温20) 洗磁珠3次,洗磁珠程序为: 加入100 μL TBST,彻底振荡混匀,置于板式磁珠收集器静置90 s,弃上清液。TBST洗磁珠3次,加入TBST稀释的 (1∶5 000) 碱性磷酸酶标记的地高辛抗体100 μL,37 ℃温育30 min。TBST洗磁珠3次,加入显色底物 (6.7 mmol∙L-1 p-NPP,0.1 mol∙L-1 Na2CO3,2 mmol∙L-1 MgCl2) 100 μL,避光显色30 min,加入2 mol∙L-1 NaOH溶液20 µL 终止显色,用酶标仪测定405 nm处的吸光值 (A405)[21,22,23,24]

结果 1 LEDGF/p75基因的合成

根据大肠杆菌密码子偏爱性优化LEDGF/p75编码基因,在5'-端添加BamH I酶切位点,在3'-端添加Sal I序列。优化后的LEDGF/p75基因序列如图 1。将合成的总长为1 605 bp的LEDGF/p75全基因序列通过T-A克隆连接到pUCm-T载体,重组载体进行DNA测序,结果表明合成序列与预期一致。将测序正确的合成序列双酶切连接到同样双酶切的表达载体pGEX-4T-1上,构建重组表达载体pGEX-4T-p75并转化DH5α感受态细胞。挑取克隆进行双酶切分析 (图 2) 和DNA序列测定。结果表明序列与预期一致,开放读码框正确,无非期待突变。双酶切验证结果见图 2

Figure 1 Sequence comparison between wt and coLEDGF/p75 genes. wt: Wild-type; co: Codon-optimized; aa: Amino acid

Figure 2 Enzyme digestion assay of recombinant expression plasmid pGEX-4T-p75. 1,2: Digested with BamH I and Sal I; M: 1 kb DNA ladder
2 宿主菌株对目的蛋白表达量的影响

10% SDS-PAGE检测结果显示在约101 kDa (因载体pGEX-4T-1具有GST标签蛋白,分子质量约为26 kDa,而LEDGF/p75分子质量约为75 kDa,因此表达的蛋白应是大约100 kDa) 处出现蛋白条带,而未诱导的培养物中未出现特异性条带。从图 3中可以看出,LEDGF/p75蛋白在宿主菌E.coli BL21和BL21 (DE3) 中均有表达,但是在BL21 (DE3) 中的表达量明显高于在E.coli BL21中的表达量,因此,选择E.coli BL21 (DE3) 作为LEDGF/p75蛋白原核表达的宿主菌。

Figure 3 10% SDS-PAGE showing expression levels of GST- co-LEDGF/p75 fusion protein with IPTG induction in E.coli BL21,BL21 (DE3),BL21 (DE3) pLysS host cells. 1: E.coli BL21/pGEX-4T-1 after induction; 2: E.coli BL21/pGEX-4T-p75 before induction; 3: E.coli BL21/pGEX-4T-p75 was induced for 2 h; 4: E.coli BL21/pGEX-4T-p75 was induced for 3 h; 5: Protein marker; 6: E.coli BL21 (DE3)/pGEX-4T-1 after induction; 7: E.coli BL21 (DE3)/pGEX-4T-p75 before induction; 8: E.coli BL21 (DE3)/ pGEX-4T-p75 was induced for 2 h; 9: E.coli BL21 (DE3)/pGEX- 4T-p75 was induced for 3 h; 10: Protein marker
3 不同诱导温度对目的蛋白表达量的影响

10% SDS-PAGE检测结果显示在约100 kDa处出现蛋白条带,而未诱导的培养物中未出现特异性条带。图 4可见,在诱导温度为30 ℃和37 ℃时,LEDGF/ p75在E.coli BL21 (DE3) 中均有表达,而诱导温度为30 ℃时,目的蛋白的表达量较高,因此,选择30 ℃作为LEDGF/p75蛋白原核表达时的诱导温度。

Figure 4 10% SDS-PAGE showing the effect of temperature on the expression level in E.coli BL21 (DE3)/pGEX-4T-p75. 1: E.coli BL21 (DE3)/pGEX-4T-1 after induction; 2: E.coli BL21 (DE3)/pGEX-4T-p75 before induction; 3,4: E.coli BL21 (DE3)/ pGEX-4T-p75 were induced at 30 ℃; 5: E.coli BL21 (DE3)/ pGEX-4T-p75 before induction; 6,7: E.coli BL21 (DE3)/pGEX- 4T-p75 were induced at 37 ℃; 8: Protein marker
4 不同诱导时间对目的蛋白表达量的影响

10% SDS-PAGE检测结果显示,蛋白诱导表达的时间分别为1、2和3 h时,目的蛋白的表达量随 诱导时间的增加而逐渐增加,而当诱导表达的时间为4 h时,目的蛋白的表达量下降,说明目的诱导表达的时间过长会导致部分降解,反倒不利于目的蛋白的积累,因此,选择3 h作为LEDGF/p75蛋白原核表达时的诱导时间 (图 5)。

Figure 5 10% SDS-PAGE showing the effect of induction time on the expression level in E.coli BL21(DE3)/pGEX-4T-p75. 1: Protein marker; 2: E.coli BL21 (DE3)/pGEX-4T-p75 before induction; 3-6: E.coli BL21 (DE3)/pGEX-4T-p75 induced for 1,2,3 and 4 h respectively; 7: E.coli BL21 (DE3)/pGEX-4T-1 after induction
5 目的蛋白的可溶性考察

10% SDS-PAGE检测结果显示,LEDGF/p75蛋白大部分以可溶的形式出现在上清液中,只有少部分存在于沉淀中 (图 6)。因此,LEDGF/p75的可溶性很好,有利于对其进行纯化。

Figure 6 10% SDS-PAGE analysis and comparison of the protein solubility for the supernatant and pellet of the induced E.coli BL21 (DE3)/pGEX-4T-p75. 1: E.coli BL21 (DE3)/pGEX- 4T-p75 before induction; 2: E.coli BL21 (DE3)/pGEX-4T-p75 after induction; 3: Supernatant of induced E.coli BL21 (DE3)/ pGEX-4T-p75; 4: Pellet of induced E.coli BL21 (DE3)/pGEX- 4T-p75; 5: Protein marker
6 LEDGF/p75蛋白的纯化

10% SDS-PAGE检测纯化后的LEDG/p75蛋白,结果显示,获得了纯化的LEDGF/p75蛋白,其纯度在90% 以上 (图 7),使用BCA法测得其浓度约为200 μg∙mL-1

Figure 7 10% SDS-PAGE analysis of protein expression and purification of induced E.coli BL21 (DE3)/pGEX-4T-p75. 1: Purified p75/LEDGF; 2: Supernatant of induced E.coli BL21 (DE3)/pGEX-4T-p75; 3: Induced E.coli BL21 (DE3)/pGEX-4T- p75; 4:E.coli BL21 (DE3)/pGEX-4T-p75 before induction; 5: Induced E.coli BL21 (DE3)/pGEX-4T-1; 6: Protein marker
7 LEDGF/p75蛋白对IN链转移反应活性的影响

活性测定设定了2个阴性对照组: 只加LEDGF/ p75,不加His-IN和LEDGF/p75。1个阳性对照: 只加His-IN。1个实验组: 同时加入His-IN和LEDGF/ p75,结果显示,只加LEDGF/p75、不加His-IN和LEDGF/p75的阴性对照组的信号值均小于0.05,只加His-IN的信号值为0.15,而同时加入His-IN和LEDGF/p75的实验组的信号达到0.3以上,是只加入His-IN的阳性对照组的2倍 (图 8)。各组数据用 SPSS 13.0软件进行方差分析,Duncan测验进行比较,发现加入His-IN的反应组与阴性对照组差异显著 (P < 0.01)。以上结果说明: 重组的HIV-1 IN具有链转移活性,而LEDGF/p75的存在能够显著提高His-IN体外催化链转移反应的活性。

Figure 8 HIV-1 IN strand transfer (STR) reaction assay with HIV IN and LEDGF/p75. IN-his: STR reaction with C-terminal His-taged IN; p75+IN-His: STR reaction with C-terminal His- taged IN and p75/LEDGF; p75: STR reaction with p75/LEDGF alone; -IN: STR reaction without IN and p75/LEDGF. (n = 5,P < 0.01)
讨论

近年来,大量参与HIV-1整合过程的宿主因子被发现[25],IN与宿主因子之间的相互作用成为抗HIV-1 IN药物筛选的新靶点,其中LEDGF/p75与IN的相互作用已经被证明是理想的抗艾滋病药物靶点[26]。随着第一代抗IN药物Raltegravir耐药性的出现,开发新一代抗整合酶药物,尤其是开发具有新的作用机制的药物迫在眉睫。本研究旨在表达LEDGF/p75酶蛋白,为研究针对LEDGF/p75-IN相互作用的HIV药物奠定基础。

本研究通过优化密码子实现了LEDGF/p75的可溶性表达。大肠杆菌表达系统是基因表达技术中发 展最早、应用最为广泛的经典表达系统,是生物技术研究和生物药物产业化进程中的重要工具[27,28,29,30]。优化表达条件的主要目的是尽可能提高目的蛋白的表达量,降低生产成本。影响外源基因表达水平的因 素很多,除了结构基因旁侧的调控元件外,基因自身的编码序列中也含有与基因表达水平相关的关键信息[31]。密码子的选择是影响基因表达的重要因素。对于同义密码子而言,不同物种的使用频率有很大差别,因此出现了所谓的密码子偏爱性。外源基因尤其是来自较远物种的基因在宿主中的表达会受到一定的影响,通过对外源基因的密码子进行优化可以提高其表达水平[32,33,34,35]。本文根据大肠杆菌的密码子偏好性,优化并全合成了人源细胞因子LEDGF/p75的编码基因,结果表明,密码子优化后的LEDGF/p75的编码在大肠菌中得到了良好的表达。通过进一步优化表达宿主菌和培养条件 (包括诱导温度和诱导时间),使得LEDGF/p75的编码基因在大肠杆菌中实现了高效可溶表达。

HIV-1的IN催化整合反应,包括3' 加工和链转移两个过程。3' 加工的产物、IN以及病毒和宿主相关蛋白因子 (包括LEDGF/p75) 形成整合前复合物(pre-integration complex,PIC),PIC在LEDGF/p75等蛋白的引导下进入细胞核,并锚定至宿主染色体,在IN的催化下完成链转移[36]。LEDGF/p75能够提高HIV-1 IN的链转移反应活性[37,38]。有研究发现,当His-tag位于HIV-1 IN的N端时,会显著降低HIV-1 IN与LEDGF/p75之间的相互作用[39],因此,本研究使用C端融合有His标签的IN来测定LEDGF/p75的功能。本文通过基于磁珠的ELISA,检测了重组p75/LEDGF对HIV-1 IN生物活性的影响,实验结果显示,在体外,LEDGF/p75确实能够提高C-端融合有His标签的HIV-1 IN的链转移反应活性,这与之前的研究结果是一致的[39,40],说明重组LEDGF/p75具有良好的生物活性,能够用于LEDGF/p75-IN相互作用的检测。为LEDGF/p75-IN相互作用抑制剂的筛选提供了良好的基础。

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