CN105793281B - 经修饰的人类酸性纤维母细胞生长因子及其组合物 - Google Patents
经修饰的人类酸性纤维母细胞生长因子及其组合物 Download PDFInfo
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Abstract
本发明提供酸性纤维母细胞生长因子(acidic fibroblast growth factor;aFGF)组合物,其包含:(i)SEQ ID NO:1的aFGF、(ii)具有N端磷酸葡糖酰化(phosphogluconoylation)的SEQ ID NO:1的aFGF、(iii)具有N端葡糖酰化(gluconoylation)的SEQ ID NO:1的aFGF、(iv)SEQ ID NO:2的aFGF及(v)SEQ ID NO:3的aFGF,或其组合。本发明亦提供经修饰的酸性纤维母细胞生长因子(aFGF),其具有N端磷酸葡糖酰化或葡糖酰化。
Description
技术领域
本发明涉及具有磷酸葡糖酰化或葡糖酰化的经修饰的酸性纤维母细胞生长因子(aFGF)。本发明亦关于aFGF组合物,其包含:(i)SEQ ID NO:1的aFGF、(ii)具有N端磷酸葡糖酰化的SEQ ID NO:1的aFGF、(iii)具有N端葡糖酰化的SEQ ID NO:1的aFGF、(iv)SEQ IDNO:2的aFGF及(v)SEQ ID NO:3的aFGF,或其组合。
背景技术
酸性纤维母细胞生长因子(aFGF),其影响各细胞类型的体外的增生及分化,是最初分离自神经组织(包括全脑及下视丘)的单链蛋白质。aFGF为肝素依赖性促分裂原(heparin-dependent mitogen)且其可强结合至所有四种已知的FGF受体及其剪接形式。其可位于与运动及感觉功能相关的神经元的特定亚群内,且可纯化自成人大脑。经纯化的aFGF为神经母细胞的促分裂原,并可促进脊髓神经元的轴突延伸。此外,aFGF经证实于培养时促进星状细胞(astrocytes)的神经生长因子表现。
数个研究报告,FGFs对神经生理活性具有范围广泛的效用,不同于其于体内中枢神经系统(central nervous system;CNS)内的促分裂作用。举例而言,当投予大脑时,aFGF可对大脑缺血所诱发的下视丘CA1神经元退化提供保护效用(Sasaki K.et al.,BrainRes.Bull.33:505-511,1994)。FGF1据报告具有保护选择性神经元群体,对抗与神经退化性疾病如阿兹海莫氏症(Guo,Z.,and Mattson,M.,Cereb.Cortex 10,50-57,2000)及HIV脑炎(Everall,I.P.et al.,J.Neuropathol.Exp.Neurol.60,293-301,2001)相关的分子的神经毒性的效用。
原生人类aFGF多肽是由154个氨基酸组成,其分离自人类大脑。然而,经确认,人类aFGF N端的19个氨基酸与人类介白素-1(interleukin-1;IL-1)具有同质性。与IL-1的氨基酸一致或类似的原生人类aFGF N端的19个氨基酸可导致相同的内源性免疫反应,包括巨噬细胞活化,以及调节细胞生长停滞(G.Venkataraman et al.,P.N.A.S.,96:3658-63,1999)。此外,据报告,促发炎性细胞激素IL-1及FGF-1(aFGF)/FGF-2(bFGF)具有相同结构骨架,且竞争酪氨酸激酶结构域的相同受体结合位(A.J.Minter et al.,J.Cell Physil.,167:229-37,1996)。
数种重组型人类aFGF于商业上可购,且广泛用于各种生物试验。举例而言,具有140个氨基酸的重组人类aFGF(R&D,aa 16-155,目录编号:232-FA/CF)、具有154个氨基酸的重组人类aFGF(R&D,aa 2-155,目录编号:231-BC/CF)、具有141个氨基酸的重组人类aFGF(Sigma,天然序列并具有额外的甲硫氨酸残基连接至N端,目录编号:F5542)。亦参见Osakada F et al.,Nat Protoc.2009;4(6):811-24,及Christina Krabbe et al.,J.Neurochem.(2009)110,1908–1920。
此外,美国专利号7,956,033(于2011发布)揭示一具有135个氨基酸的经修饰的人类aFGF,其呈现增进的稳定性,包含通过删除原生人类aFGF N端的20个氨基酸的缩短的原生人类aFGF,并于缩短的原生aFGF之前加上丙胺酸(Alanine;Ala)。
发明内容
在一方面,本发明提供酸性纤维母细胞生长因子(aFGF)组合物,其包含:(i)SEQID NO:1的aFGF、(ii)具有N端磷酸葡糖酰化的SEQ ID NO:1的aFGF、(iii)具有N端葡糖酰化的SEQ ID NO:1的aFGF、(iv)SEQ ID NO:2的aFGF及(v)SEQ ID NO:3的aFGF,或其组合。
在本发明的一具体实施例中,aFGF组合物具有下列高效能离子交换层析(highperformance ion-exchange chromatography;HPIEC)尖峰:
尖峰 | 滞留时间(min) |
1 | 18.2 |
2 | 18.5 |
3 | 19.1 |
4 | 19.4 |
5 | 19.9 |
其中该HPIEC于下列条件进行:
流速:0.5mL/min
样本量:100μL
管柱:ProPac离子交换管柱
吸光值:220-280nm
溶析设定:
缓冲液A:20mM MES,pH 6.0-8.0
缓冲液B:20mM MES+1M NaCl,pH 6.0-8.0
时间(min) | 缓冲液A(%) | 缓冲液B(%) |
0 | 95 | 5 |
5 | 95 | 5 |
15 | 45 | 55 |
25 | 40 | 60 |
30 | 20 | 80 |
40 | 95 | 5 |
另一方面,本发明提供经修饰的酸性纤维母细胞生长因子(aFGF),其具有N端磷酸葡糖酰化或葡糖酰化。
根据本发明,相较于SEQ ID NO:1的人类aFGF或野生型人类aFGF,该aFGF组合物及经修饰的aFGF呈现增进的特异活性。
附图说明
配合附图阅读可更佳地理解前文的概述以及如后的本发明的详细说明。在附图中:
图1显示aFGF135(SEQ ID NO:1)样本的HPIEC轮廓。
图2A-图2C提供aFGF135、aFGF140、及aFGF154的HPIEC轮廓。图2A显示aFGF135的HPIEC轮廓、图2B显示aFGF140的HPIEC轮廓、及图2C显示aFGF154的HPIEC轮廓。
图3显示aFGF135样本的HPIEC轮廓的五个尖峰样本的特异活性。
具体实施方式
除非另行定义,本文使用的所有技术性及科学性术语具有与本发明所属领域中的本领域技术人员一般所理解相同的意义。
除非另有明确指明,本文使用的单数形式“一”、及“该”包括复数指涉物。因此,举例而言,提及“一样本”时是包含复数个此类样本及本领域技术人员已知的其等同物。
本文所使用的“酸性纤维母细胞生长因子”或“aFGF”术语是指肝素依赖性促分裂原,且其可强结合至所有四种已知的FGF受体及其剪接形式,包括任何类型的aFGFs,优选为人类aFGF,其可为原生、野生型、或经修饰的aFGF。在一优选具体实施例中,该aFGF具有SEQID NO:1的氨基酸序列,如美国专利号7,956,033所揭示的,其在此并入本申请作为参考数据。
本发明意外发现,相较于无磷酸葡糖酰化及葡糖酰化的aFGF、或野生型aFGF,具有N端磷酸葡糖酰化或葡糖酰化的经修饰的aFGF呈现增进的特异活性(specific activity)。
在本发明的一具体实施例中,aFGF具有SEQ ID NO:1的氨基酸序列。
在本发明的一优选实例中,本发明的经修饰的aFGF是通过将磷酸葡糖酰化作用或葡糖酰化作用加至aFGF而进行,该aFGF优选为人类aFGF,最优选为SEQ ID NO:1的人类aFGF。本发明意外发现,经修饰的人类aFGF具有优异的特异活性。
本文所使用的“磷酸葡糖酰化”术语是指通过添加一个或多个6-磷酸葡糖酰基以修饰或化学修饰一分子(如蛋白质)。
本文所使用的“磷酸葡糖酰化”术语是指通过添加一个或多个磷酸葡糖酰基以修饰或化学修饰一分子(如蛋白质)。在本发明的一具体实施例中,aFGF的磷酸葡糖酰化的进行是通过将一6-磷酸葡糖酰基加至aFGF。
本文所使用的“葡糖酰化”术语是指通过添加一或多个葡糖酰基以修饰或化学修饰一分子(如蛋白质)。在本发明的一实例中,aFGF的葡糖酰化的进行是通过将一6-葡糖酰基加至aFGF。此外,根据本发明,具有葡糖酰化的aFGF可来自具有磷酸葡糖酰化的aFGF的去磷酸化作用(dephosphorylation)。根据本发明的另一实例,aFGF的葡糖酰化的进行是通过将6-磷酸葡糖酰基加至aFGF并随后去磷酸化。根据本发明,相较于无磷酸葡糖酰化及葡糖酰化的aFGF、或野生型aFGF,具有葡糖酰化的经修饰的人类aFGF与具有磷酸葡糖酰化的经修饰的人类aFGF两者具有增进的特异活性。
蛋白质的磷酸葡糖酰化或葡糖酰化可经由天然过程或化学修饰(如合成方法)进行。另一方面,由于大肠杆菌(Escherichia coli;“E.coli”)表现的蛋白质可能出现葡糖酰化或磷酸葡糖酰化(如α-N-6-磷酸葡糖酰化)等转译后修饰(Geoghegan,et al.,Anal.Biochem.267:169-184(1999);Kim et al.,Acta Crystallographica Section D-Biological Crystallography 57:759-762(2001);Yan,et al.Biochemical&BiophysicalResearch Communications262:793-800(1999)“Yan et al.I”;Yan,et al.,Biochemical&Biophysical Research Communications 259:271-282(1999)“Yan et al.II”),可通过在适当大肠杆菌菌株表现,以产生经磷酸葡糖酰化或经葡糖酰化的蛋白质。
据报告,此种转译后修饰可能对所表现的蛋白质的活性、稳定性、结构或免疫原性产生不良影响。然而,本发明令人惊讶地发现,相较于无磷酸葡糖酰化与葡糖酰化的人类aFGF,或野生型人类aFGF,磷酸葡糖酰化或葡糖酰化的人类aFGF呈现增进的特异活性。在一具体实施例中,该人类aFGF为SEQ ID NO:1的人类aFGF。
本文所使用的“aFGF特异活性”或“特异活性”是指单位量的样本的一aFGF活性,其是由生物试验测定。
另一方面,本发明提供一酸性纤维母细胞生长因子(aFGF)组合物,其包含:(i)SEQID NO:1的aFGF、(ii)具有N端磷酸葡糖酰化的SEQ ID NO:1的aFGF、(iii)具有N端葡糖酰化的SEQ ID NO:1的aFGF、(iv)SEQ ID NO:2的aFGF及(v)SEQ ID NO:3的aFGF,或其组合。
SEQ ID NO:2的aFGF为经修饰的SEQ ID NO:1的aFGF,其是以赖氨酸取代位置101的天冬酰胺酸。SEQ ID NO:3的aFGF为经修饰的SEQ ID NO:1的aFGF,其是以赖氨酸取代位置2的天冬酰胺酸。
在本发明的一具体实施例中,aFGF组合物是由:(i)SEQ ID NO:1的aFGF、(ii)具有N端磷酸葡糖酰化的SEQ ID NO:1的aFGF、(iii)具有N端葡糖酰化的SEQ ID NO:1的aFGF、(iv)SEQ ID NO:2的aFGF、及(v)SEQ ID NO:3的aFGF,或其组合所组成。
在本发明的一具体实施例中,该aFGF组合物具有下列HPIEC尖峰:
尖峰 | 滞留时间(min) |
1 | 18.2 |
2 | 18.5 |
3 | 19.1 |
4 | 19.4 |
5 | 19.9 |
其中该HPIEC于下列条件进行:
流速:0.5mL/min
样本量:100μL
管柱:ProPac离子交换管柱
吸光值:220-280nm
溶析设定:
缓冲液A:20mM MES,pH 6.0-8.0
缓冲液B:20mM MES+1M NaCl,pH 6.0-8.0
时间(min) | 缓冲液A(%) | 缓冲液B(%) |
0 | 95 | 5 |
5 | 95 | 5 |
15 | 45 | 55 |
25 | 40 | 60 |
30 | 20 | 80 |
40 | 95 | 5 |
相较于SEQ ID NO:1的人类aFGF或野生型人类aFGF,本发明的aFGF组合物显示增进的特异活性。
本发明进一步以下列实例说明,其提供的目的在于例示而非限制。
实施例
实施例1:经修饰的SEQ ID NO:1的人类aFGF(“aFGF135”)的表现及分离
构建一载体pET3c-haFGF以表现aFGF135,如美国专利号7,956,033所描述,其所揭示的在此全部并入本申请作为参考数据。
扩增pET3c-haFGF并随后转形至大肠杆菌BL21(DE3)(Novagen,Germany)胜任细胞。在以IPTG诱导之前,抗安比西林的大肠杆菌菌落是于LB培养基培养及扩增至OD600=0.3。培养16小时(±2小时)之后,收获细菌并以5000rpm离心以移除上清液。收获的细菌以PBS清洗二次,随后以高压均质机溶裂;接着流经孔径大小0.22μm的滤膜。经过滤的培养基可用于蛋白质分离。
该aFGF135的胜肽是以三种层析管柱分离:(1)阳离子交换层析;(2)亲和力层析;以及(3)粒径排阻层析。上述管柱所使用的缓冲液为磷酸盐溶液(Na2PO4:NaHPO3=51:49,并具0.1%EDTA-Na,pH 6.0-8.0)。
实施例2:表现的蛋白质的HPIEC(高效能离子交换层析)
将如实施例1所获得的经分离的胜肽组合物进行HPIEC分析。层析条件如下:移动相:缓冲液A-20mM MES,pH 6.0-8.0,缓冲液B-20mM MES+1M NaCl,pH 6.0-8.0;管柱:ProPac离子交换管柱;吸光值:220-280nm;注射体积(样本量):100μL;流速:0.5mL/min;时间:45min.;以及温度:25℃,其中梯度曲线为:
时间(min) | 缓冲液A(%) | 缓冲液B(%) |
0 | 95 | 5 |
5 | 95 | 5 |
15 | 45 | 55 |
25 | 40 | 60 |
30 | 20 | 80 |
40 | 95 | 5 |
Balb/3T3细胞增生试验的进行如下述。选择生长良好的Balb/3T3细胞并计算细胞浓度。试验使用96孔培养盘,其中60孔(B2至G11)加入试验样本,且其他周围的孔加入无菌PBS。细胞以每孔2x 104个细胞的密度种植于培养盘,其中培养基为RPMI-1640+10%FBS,并于37℃、5%CO2下培养24±2小时。制备RPMI-1640培养基+0.5%FBS的样本稀释缓冲液。以样本稀释缓冲液(RPMI-1640培养基+0.5%FBS)将实施例1取得的标准aFGF组合物稀释成16个单位/ml,并连续稀释2倍浓度,以取得至少经6个连续稀释的样本。随后,移除96孔培养盘的培养基,将100μL的样本加入各孔,并于37℃、5%CO2下培养24±2小时。针对活性测定,首先将培养盘置于室温下15分钟,接着将100μL的发光试剂加至各孔反应30分钟。最后,检测发光信号,并以并行线分析法(Parallel-Line Assay)计算活性。
结果显示于图1及下表1。分别于滞留时间(min.)约18.165、18.504、19.131、19.419、及19.884时观察到5个主要尖峰。
表1:
实施例3:不同人类aFGFs的HPIEC轮廓的比较
本实施例中测试三种不同的人类aFGF,即aFGF135、aFGF140、及aFGF154。SEQ IDNO:1的aFGF135的制备如前实施例1所述。aFGF140(具有140个氨基酸)为R&D重组酸性人类FGFaa 16-155(目录编号:232-FA/CF),其购自R&D Systems,Inc.,Minneapolis,MN55413USA。aFGF154(具有154个氨基酸)为R&D重组酸性人类FGF aa 2-155(目录编号:231-BC/CF),其购自R&D Systems,Inc.,Minneapolis,MN55413USA。层析条件与前文实施例2所述相同。
结果如图2A-图2C所示。图2A显示aFGF135的HPIEC轮廓、图2B显示aFGF140的HPIEC轮廓、及图2C显示aFGF154的HPIEC轮廓。如图2A-图2C所示,aFGF135、aFGF140、及aFGF154具有极不同的HPIEC轮廓。
实施例4:以LC-MS/MS确认aFGF135的HPIEC尖峰并比较其特异活性
1.Arg-C切割:首先以3kDa截留(cut-off)的Amicon超离心过滤器(Amicon UltraCentrifugal Filter)浓缩各尖峰的样本。所有16个样本(10μg,取自四批次的HPIEC尖峰1、2、4、及5)以50mM碳酸氢铵缓冲液稀释至100μL。接着,于37℃下,所有蛋白质样本以6M尿素变性并以1μL 1M DTT还原1小时。于室温避光下,以10μL 0.5M IAM进行30min烷化。所得的haFGF蛋白质以50mM ABC缓冲液稀释,随后于37℃下以0.5μg Arg-C(蛋白质:酵素=20:1)切割17小时。于切割后,所有样本以Zip Tip(Millipore)脱盐、以真空离心(speedvac)干燥、及以0.1%FA重新溶解,以进行LC-MS/MS分析。
2.LC-MS/MS分析:以Q-Exactive质谱仪连接Ultimate 3000RSLC系统,分析经切割的样本(0.5μg)。以C18管柱(Acclaim PepMap RSLC,75μm x150mm,2μm,)进行液相层析(LC)分离,其中梯度显示如下:
时间(min) | A% | B% | 流速(μL/min) |
0 | 99 | 1 | 0.25 |
7 | 99 | 1 | 0.25 |
47 | 60 | 40 | 0.25 |
55 | 10 | 90 | 0.25 |
65 | 10 | 90 | 0.25 |
70 | 99 | 1 | 0.25 |
75 | 99 | 1 | 0.25 |
(移动相A:5%ACN/0.1%FA;移动相B:95%ACN/0.1%FA)
进行全MS扫描,其范围为m/z 380-1800,并将MS扫描中的10个最强离子进行碎断(fragmentation),以用于MS/MS光谱。通过Proteome Discoverer1.4,将原始数据处理成尖峰列表,以进行Mascot数据库搜寻。
3.数据库搜寻参数:以Mascot 2.4.0进行数据库搜寻。所使用的参数如下:
数据库:由供货商提供的序列
酵素:Arg-C*
可变修饰:磷酸葡糖酰化(蛋白质N端);葡糖酰化(蛋白质N端);甲基化(Asn、Ala、及Lys)
胜肽质量公差(mass tolerance):±10ppm
碎片质量公差:±0.02Da
最大错失切割(Max missed cleavages):5
仪器类型:ESI-TRAP
离子截留(cut-off)值:30
Arg-C*:此酵素已被证明切割精胺酰基残基的羧基端、Lys-Lys键与Lys-Arg键。
3.结果:
表2:LC-MS/MS结果
“位置”表示haFGF的残基编号。“观察值”表示探测扫描所观察到的m/z值,而“Mr(实验值)”、“Mr(计算值)”、“ppm”分别代表实验分子量(molecular weight;MW)、MW计算值、及观察MW与理论MW间的差异。MC表示错失切割。计值(scores)为直接来自Mascot数据库搜索引擎的胜肽离子分数。
经由LC-MS,发现尖峰3的样本为无修饰的aFGF135。
表3:aFGF的修饰
此外,比较5个尖峰的样本的特异活性。进行Balb/3T3细胞增生生物试验,其方法如前实施例2所述。如图3所示,相较于尖峰3、4、或5的样本,尖峰1与2的样本呈现较高的特异活性。
据信,本发明所属领域中的本领域技术人员基于本文的描述,无须进一步的说明,可最大限度利用本发明。因此,所提供的说明及权利要求书应理解为例示的目的,而非以任何方式限制本发明的范围。
Claims (3)
1.一种经分离的酸性纤维母细胞生长因子(aFGF),其氨基酸序列由SEQ ID NO:1所组成,且其N端经磷酸葡糖酰化(phosphogluconoylated),其特征是其高效能离子交换层析(high performance ion-exchange chromatography;HPIEC)在滞留时间18.2分钟有一个尖峰,其中该HPIEC系于下列条件进行:
流速:0.5mL/min
样本量:100μL
管柱:ProPac离子交换管柱
吸光值:220-280nm
溶析设定:
缓冲液A:20mM MES,pH 6.0-8.0
缓冲液B:20mM MES+1M NaCl,pH 6.0-8.0
。
2.一种经分离的酸性纤维母细胞生长因子(aFGF),其氨基酸序列由SEQ ID NO:1所组成,且其N端经葡糖酰化(gluconoylated),其特征是其高效能离子交换层析(highperformance ion-exchange chromatography;HPIEC)在滞留时间18.5分钟有一个尖峰,其中该HPIEC系于下列条件进行:
流速:0.5mL/min
样本量:100μL
管柱:ProPac离子交换管柱
吸光值:220-280nm
溶析设定:
缓冲液A:20mM MES,pH 6.0-8.0
缓冲液B:20mM MES+1M NaCl,pH 6.0-8.0
。
3.根据权利要求1或2所述的经分离的aFGF,相较于SEQ ID NO:1的人类aFGF或野生型人类aFGF,其具有增进的特异活性。
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