李雪芹候蓓蓓赵军田明玉修志龙*
连理工学环境生命学院 连 116024
摘:目 水蛭素血浆半衰期短严重限制床应聚乙二醇修饰效延长半衰期文通拟修饰位点修饰方法单修饰产物率纯度活性保存率修饰专性强活性保存率较高修饰策略方法采液相固相修饰方法琥珀酰亚胺活化PEG 20kDa分pH6080条件水蛭素HisLys进行定点修饰SDSPAGE分析产物修饰度离子交换柱修饰产物进行离然凝血酶滴定法测定水蛭素单修饰产物体外抗凝活性外分子动力学模拟方法预测pH80条件PEG修饰位点结果 pH6080条件水蛭素单修饰率高达90二者单修饰活性保存率相差较pH60时液固相单修饰活性保存率34348pH80时分5596结 pH80 条件采 离子交换柱辅助〞固相修饰方法Lys进行定点修饰较高产率较高活性保存率单修饰产物
关键词:重组水蛭素SCmPEG抗凝活力分子动力学模拟
PEGylation of Recombinant Hirudin
Li XueqinHou BeibeiZhao JunTian MingyuXiu Zhilong*
Department of Bioscience and BiotechnologySchool of Environmental and Biological Science and TechnologyDalian University of TechnologyDalian 116024China
Abstract OBJECT Hirudin is the most potent inhibitor of thrombin found in nature Although hirudin has the strongest antithrombin activity in vivo its short halflife in serum significantly limits its clinical anticoagulant application Currently PEGylation is commonly used as an effective method to prolong its halflife in serum Our object of experiment is to choose the best PEGylation method according to the monoPEGylated recombinant hirudin’s purity and the anticoagulant activity in vitro METHODS Solution method and solid method assisted by packedbed〞 and anion exchange column were used to favor the formation of monoPEGylated hirudin The mild acidic and mild alkaline PEGylation strategy was used to target His residue and Lys residue of hirudin respectively using SCmPEG 20kDa The pegylated products were isolated by anion exchange chromatogram SDSPAGE was used to analyze the purity of PEGylated hirudin Thrombin method was used to analyze the anticoagulant activity of PEGylated hirudin Molecular dynamics modeling was used to judge the probability of PEGylation site RESULTS The monoPEGylated product with a purity of more than 90 was obtained at pH60 and pH80 But a large difference in anticoagulant activity exists the anticoagulant activity of solution and solid methods were 34 and 348 respectively at pH605596 at pH80 CONCLUSION Solid method assisted by anion exchange column at pH80 was a better strategy to get monoPEGylated rHirudin
Key words:recombinant hirudinSCmPEGanticoagulant activitymolecular dynamics modeling
前言
水蛭素含65氨基酸残基3二硫键肽分子量约7000迄止发现特异性凝血酶抑制剂[1]凝血酶诱发血液凝固诱导血血栓形成重原水蛭素种血栓病均疗效
然水蛭素显著药缺点血浆半衰期较短般60100分钟患者需断注射维持抗凝效果导致治疗钱较高重复注射会引起良反响[24]世纪70年代发现蛋白PEG修饰方面药特性改善开展水蛭素PEG修饰研究George CAvgerinos[5]等采基工程方法水蛭素氨基酸进行改造甲基营养酵母 Hansenyla polymorpha特异性表达含两Lys水蛭素采硝基碳酸酯活化PEG 5kDa进行修饰设计工业等级纯化步骤种水蛭素修改方式提高修饰产物专性研究周期性较长须具备定科研条件
国关水蛭素PEG修饰研究爱[6]等采SPAPEG 5kDa修饰水蛭素II采Source Q15离子交换柱凝胶柱离修饰产物发现三修饰产物活力降低原蛋白335秦海娜[7]等采羰基二咪唑法活化PEG 5kDa修饰水蛭素采凝胶色谱方法崔修饰产物进行离水蛭素修饰产物离修饰产物间未离开
实验组旨通拟修饰位点修饰方法单修饰产物率纯度活性保存率找修饰专性强活性保存率较高修饰策略修饰位点选择HisLys修饰方法选择液相修饰 填料辅助〞离子交换柱辅助〞两种固相修饰方法
1 材料
基工程重组水蛭素〔rHir〕购连高新生物制药水蛭素变异体2购重庆科润生物医药SCmPEG 20kDa购北京键凯科技
2 实验方法
21 水蛭素His位点PEG修饰
211液相修饰
HV2SCmPEG 20 kDa摩尔 11 溶02M pH60磷酸盐缓溶液中〔PBS〕25℃中反响15h反响产物HiTrap Q HP进行线性梯度洗脱
填料辅助〞固相修饰
pH6020mM PBSrHir SCmPEG摩尔13QSepherose FF介质中充分反响反响完成装柱梯度洗脱
22 水蛭素Lys位点PEG修饰
221 液相修饰
HV2SCmPEG20 kDa摩尔 13 溶002 M pH80 PBS中23℃反响SCmPEG分三等分三次参加次反响30min反响结束样品立HiTrap Q HP进行线性梯度洗脱
222 离子交换柱辅助〞固相修饰
4 ml 15mgml HV2 溶液 (溶PBS 20 mM pH 8 A液〕溶4 ml 20 mM pH 8 PBS 中 SCmPEG先1 mlmin流速样柱中二者柱中充分反响进行线梯度洗脱
3 分析方法
31 SDS–PAGE
参考文献[8]15浓缩胶4离胶采含5氯化钡01M碘液PEG进行染色表1水蛭素PEG修饰产物理分子量结合电泳图确定洗脱组分成分
表1 水蛭素PEG修饰产物组分理分子量
Table 1 The estimated molecular weights of the PEGylated hirudin
32 生物活性测定
参文献[9]方法采凝血酶滴定法
33 组氨酸修饰产物含量检测
利SCmPEGrHirHis残基间形成氨基甲酸酯键中性羟胺敏感性测定组氨酸修饰位置异构体含量
34 Lys修饰位点预测
采溶剂外表积作判定赖氨酸残基修饰性判断标准运分子动力学模拟方法分析预测液相固相修饰位点
4 结果
41 水蛭素His位点PEG修饰
411 液相修饰
4 修饰产物离纯化
液相方法制备PEG化水蛭素采离子交换柱进行离纯化SDSPAGE法进行组分鉴定
图1 阴离子交换色谱离修饰反响混合物〔pH60〕
Fig1 Separation of the PEGylation mixture (prepared at pH60) by anionexchange
chromatography
图2 修饰产物电泳结果〔155〕
Fig 2 SDSPAGE analysis of PEGylated rhirudin
结合表1图2推测出图1中峰 12 3分二修饰产物单修饰产物单修饰产物通积分法计算出图1中峰面积出出产物例:峰1:峰2:峰3 474:946:066见单修饰产物修饰产物
4 单修饰产物体外抗凝活性测定
采凝血酶滴定法修饰水蛭素进行活性检测
图3 重组水蛭素单修饰重组水蛭素体外抗凝活性测定
Fig 3 In vitro anticoagulant activity of rhirudin and monopegylated rhirudin
图3显示峰2相未修饰rhirudin抗凝活性保存率34 峰3抗凝活性保存率192峰2具较高抗凝活性保存率修饰反响产物〔峰2占修饰反响产物946〕选作文目标单修饰产物做进步分析
4 His修饰位置异构体含量测定
利SCmPEGrHirHis残基间形成氨基甲酸酯键中性羟胺敏感性测定组氨酸修饰位置异构体含量
图4 组氨酸修饰位置异构体含量测定
Fig 4 The proportion assay of Hispegylated positional isomer
图4中峰27971中性羟胺断开rHirdin 说明His修饰位置异构体含量797102M pH60 PBS中修饰反响成份
412 填料辅助〞固相修饰
4121 修饰产物离纯化
pH60 20mM PBS rHir:SCmPEG1:3反响固相修饰产物采阶段洗脱策略进行离SDSPAGE法进行组分鉴定
图5 固相修饰反响混合物〔pH60 20mM PBS rHir SCmPEG13〕离纯化
Fig 5 Separation of the solidphase assisted PEGylation reaction mixture (pH60
20mM rHir SCmPEG13 )
图6 固相修饰反响混合物(pH60 20mM PBSrHirSCmPEG13)纯化产物电
泳图谱
Fig 6 SDSPAGE analysis of the separation products of the solidphase assisted
PEGylation reaction mixture (pH60 20mM PBS rHir SCmPEG13 )
图5示峰1盐浓度突然增造成紫外吸收波动峰2电泳检测图6示分子量约27KDa推断单修饰产物峰3高效凝胶滤检测具标准重组水蛭素相洗脱体积推断未反响水蛭素
4122 His修饰位置异构体含量测定
利中性羟胺法修饰位置异构体含量进行检测
图7 固相辅助单修饰产物高效凝胶滤图谱
Fig 7 HPSEC analysis of the monoPEGylated product of the solidphase assisted
PEGylation
图7知中性羟胺作未翻开mPEG重组水蛭素间连接键修饰反响未发生His51重组水蛭素中mPEGSC发生修饰氨基酸残基Lysε氨基N末端氨基文献报道N末端氨基pKa 78Lysε 氨基pKa 101[10]着pH降低N末端氨基反响活性Lysε 氨基[1112] pH60条件反响活性较高推测修饰反响发生N末端氨基
单修饰产物体外抗凝活性测定
采凝血酶滴定法修饰产物进行活性检测
表2 凝血酶滴定法测固相辅助单修饰产物活性
Table 2 Analysis of the monoPEGylated product of solidphase assisted PEGylation
by thrombin titration method
表2知单修饰产物抗凝活性原蛋白〔rHir〕3485该修饰产物活性保存率略高液相修饰反响单修饰产物活性〔34〕pH6020mMPBS液相修饰反响中7971单修饰产物修饰位点组氨酸残基相反响条件固相辅助修饰单修饰产物修饰均未发生组氨酸两种单修饰产物活性差异原
42 水蛭素Lys位点PEG修饰
421 液相修饰产物离纯化
采离子交换柱SCmPEG 20 kDa修饰产物进行离纯化SDSPAGE法离组分进行鉴定
图8 SCmPEG 20kDa液相修饰产物离子交换色谱图
Fig 8 Ion Exchange Chromatography of the SCmPEG 20kDa HV 2 prepared in
solution phase
图9 SCmPEG 20kDa液相修饰产物离子交换洗脱物电泳图
Fig9 SDSPAGE of peaks collected from Fig8
结合表1图9知图8中峰 1 2分二修饰单修饰产物通积分法计算出图8中峰面积出产物例:峰1峰2 97903见单修饰产物修饰产物
422 离子交换柱辅助〞固相修饰产物离纯化
离子交换柱辅助〞固相修饰方法产物采阶段洗脱策略进行离SDSPAGE法进行组分鉴定
图10 SCmPEG 20kDa固相辅助修饰产物离子交换色谱图
Fig10 Ion Exchange Chromatography of the SCmPEG 20kDa HV 2 on column
modification
图11 SCmPEG 20kDa固相辅助修饰产物离子交换洗脱物电泳图
Fig 11 SDSPAGE of peaks collected from Fig10
洗脱组分鉴定421图10中峰12分SCmPEG 20kDa二修饰单修饰产物通积分法出图10中产物例:峰1:峰2 39:961相液相离纯度修饰专性显著提高图811拟中发现固相修饰转化率液相低固相修饰中柱填充着离子交换介质导致环境复杂蛋白PEG水溶液中状态分子差异柱中分配致反响物接触率降低
423 修饰产物体外抗凝活力测定
采凝血酶滴定法修饰水蛭素进行活性检测
图12 种单修饰水蛭素体外活
Fig 12 The specific activity ratios of the monoPEGylated HV2s in vitro
图12示固相修饰蛋白活力保存率显著高液相修饰固相修饰提高修饰反响单性活力保存率拟高种现象角度说明固相修饰提高修饰专性
水蛭素修饰位点预测分析
采溶剂外表积法水蛭素修饰位点预测发现:液相修饰中Lys47外三赖氨酸NH2残基拟容易修饰固相修饰中Lys 27 Lys 35Lys 24 Lys 47更易修饰Lys27位N域核心部位三二硫键PEGK27修饰会降低水蛭素活性Lys35位水蛭素柔性伸溶液环远离水蛭素凝血酶作区远离水蛭素N域核心推测活性较高PEG修饰产物应该修饰Lys35
5 讨
目前修饰蛋白质活化PEG种类较根蛋白质身特点求选择种类活化PEG例专性修饰肽链N端氨基半胱氨酸巯基赖氨酸侧链氨基等[13]选择修饰基团必须蛋白质功非必需基团否导致蛋白质活性降甚完全丧失文选择位非活性中心HisLys进行修饰
〔1〕pH60条件SCmPEG 20kDaHis位点进行定点修饰液相修饰中组氨酸位置异构体单修饰产物目单修饰产物占修饰产物946 微酸性条件修饰产物目单修饰产物体外抗凝活性保存率34 填料辅助〞固相修饰中pH60 20mM PBS rHir SCmPEG13条件单修饰产物抗凝活性保存率达3485通检测修饰产物中SCmPEG重组水蛭素偶联键检测推断修饰位点N末端氨基
〔2〕pH80条件SCmPEG 20kDaLys位点进行定点修饰采分子动力学模拟法预测:液相修饰位点Lys 27Lys 35Lys 24Lys35固相修饰修饰位点实验条件液相修饰离子交换柱辅助〞固相修饰中单修饰产物分占修饰产物903961活性保存率分5596固液相修饰专性纯度微酸条件修饰水相活性保存率显著提高固相修饰明显优液相修饰pH80 条件采 离子交换柱辅助〞固相修饰方法lys进行定点修饰较高率纯度较高活性保存率单修饰产物
实验实现修饰反响离偶联减少操作步骤离子交换辅助固相修饰方法水蛭素单修饰开辟新途径存缺乏固相修饰法转化率较低活化PEG易水解等问题尝试通离子交换填料进行分析研究
参考文献:
[1]MarkwartFThe development of hirudin as an antithrombotic drug〞ThrombRes741—23(1994)
[2]RydelTJRavichandranKGTulinskyABodeWHuberRRoitschCFentonJWThe structure of a complex of recombinant hirudin and humanαthrombin〞Science249277—280(1990)
[3]RydelTJTulinskyABodeWHuberRRefined structure of the hirudinthrombin complex〞JMolBiol221583—601(1991)
[4]MarkwardtFPastpresent and future of hirudin〞Haemost 21(Suppl1)11—26(1991)
[5]George C A Brian G T Kenneth J G et al Production and Clinical
Development of a Hansenula polymorphaDerived PEGylated Hirudin Seminars
in Thrombosis and Hemostasis 2001 357371
[6]爱蒋中华钟根深董春娜吴祖泽聚乙二醇修饰水蛭素离纯化活性分析药物生物技术2004 11 (5) 30205
[7]Haina QinZhiLong XiuDalian Zhang et al Improved Analytical Methods for Pegylated HirudinChinese J Chen Eng200715(4)586590
[8] Stepaniants S Izrailev S and Schulten K Extraction of lipids from phospholipid membranes by steered molecular dynamics J Mol Model 1997 3473– 475
[9] Vitali J Martin PD Malkowski MG et al J Biol Chem 1992 26717670
[10]Markwarkt F Hirudin as inhibitor of thrombin In SPColowick NOKaplan editiors In Methods in Enzymology 1th ed New York Academic Press 924321957
[11]Wong S S Chemistry of protein conjugation and crosslinking 1991CRC Press Boca Raton FL
[12]Lee H Jang I H Ryu S H and Park T G Nterminal sitespecific monoPEGylation of epidermal growth factor PharmRes 200320818825
[13]RobertsMJ BentleyMD Harris JM Chemistry for peptide and protein pegylation[J]Adv Drug Deliv Rev 2002 54(4) 45947
附图表
表1 水蛭素PEG修饰产物组分理分子量
Table 1 The estimated molecular weights of the PEGylated hirudin
PEGylated products
MWkDa
DiPEGylated(20 kDa)HV2
47
Mono PEGylated(20 kDa)HV2
27
Tri PEGylated(5 kDa)HV2
22
Di PEGylated(5 kDa)HV2
17
Mono PEGylated(5 kDa)HV2
12
表2凝血酶滴定法测固相辅助单修饰产物活性
Table 2 Analysis of the monoPEGylated product of
solidphase assisted PEGylation by thrombin titration method
滴定样品
耗标准酶活单位
样品浓度〔mgml〕
单修饰产物
115v
0011
rHir()
30v
0001
图1 阴离子交换色谱离修饰反响混合物〔pH60〕
Fig1 Separation of the PEGylation mixture (prepared at pH60) by anionexchange chromatography
图2 修饰产物电泳结果〔155〕
Fig2 SDSPAGE analysis of PEGylated rhirudin
注:1标准PEGs2图1中峰1 3:图1中峰2 4 图1中峰3
图3 重组水蛭素单修饰重组水蛭素体外抗凝活性测定
Fig3 In vitro anticoagulant activity of rhirudin and monopegylated rhirudin
图4 组氨酸修饰位置异构体含量测定
Fig 4 The proporton assay of Hispegylated positional isomer
注:1液相单修饰产物〔图1中峰2 中性羟胺处理〕
2液相辅助单修饰产物〔图1中峰2〕
3 rHirudin()
图5 固相修饰反响混合物〔pH60 20mM PBS rHir SCmPEG13〕离纯化
Fig5 Separation of the solidphase assisted PEGylation reaction mixture (pH60 20mM PBS rHir SCmPEG13 )
图6 固相修饰反响混合物(pH60 20mM PBSrHirSCmPEG13)纯化产物电泳图谱
Fig6 SDSPAGE analysis of the separation products of the solidphase assisted PEGylation reaction mixture (pH60 20mM PBS rHir SCmPEG13 )
注:1:标准PEGs2:图5中峰13:图5中峰24:图5中峰3
图7 固相辅助单修饰产物高效凝胶滤图谱
Fig7 HPSEC analysis of the monoPEGylated product of the solidphase assisted PEGylation
注:1固相辅助单修饰产物〔图5中峰2 中性羟胺处理〕
2固相辅助单修饰产物〔图5中峰2〕
3图5中峰3
4rHirudin()
图8 SCmPEG 20kDa液相修饰产物离子交换色谱图
Fig 8 Ion Exchange Chromatography of the SCmPEG 20kDa HV 2 prepared in solution phase
35kDa
20kDa
12kDa
10kDa
Marker Peak1 Peak2
图9 SCmPEG 20 kDa液相修饰产物离子交换洗脱物电泳图
Fig9 SDSPAGE of peaks collected from Fig8
图10 SCmPEG 20kDa固相辅助修饰产物离子交换色谱图
Fig10 Ion Exchange Chromatography of the SCmPEG 20kDa HV 2 on column modification
Marker Peak1 Peak2
35kDa
20kDa
12kDa
10kDa
图11 SCmPEG 20kDa固相辅助修饰产物离子交换洗脱物电泳图
Fig11 SDSPAGE of peaks collected from Fig10
图12 种单修饰水蛭素体外活
Fig12 The specific activity ratios of the monoPEGylated HV2s in vitro
1 SCmPEG 20kDa液相修饰产物2 SCmPEG 20kDa固相修饰产物
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