N-端聚乙二醇化L-门冬酰胺酶的制备、纯化及鉴定研究
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摘要
左旋门冬酰胺酶(L-ASP)是一种白色或微黄色冻干粉未,无嗅、无味,由4个亚基组成的同型四聚体,其相对分子量为133000±5000,主要作用是把血清中的门冬酰胺水解为门冬氨酸和氨,临床上主要用于治疗急性淋巴细胞性白血病(简称急淋)。急性白血病等肿瘤细胞会因既不能从血中取得足够门冬酰胺,亦不能自身合成,使其蛋白质合成受障碍,增殖受抑制,细胞大量破坏而不能生长、存活;正常细胞有自身合成门冬酰胺的功能而不受影响。本品亦能干扰细胞DNA、RNA的合成,可能作用于细胞G1增殖周期中。在临床应用上,重组L-ASP与其它的重组制品一样具有半衰期短、有较强的免疫原性、毒副作用大等缺点。由于体内半衰期较短,需要每天注射才能维持有效的血药浓度。结果长期而频繁的注射,增加了治疗成本,给患者带来严重的痛苦和不便,降低了依从性,加重了病人的经济负担。
为了克服L-ASP在临床用药上的限制,本课题选择单甲氧基聚乙二醇丙醛(mPEG-ALD)对左旋门冬酰胺酶(L-ASP)进行N-末端定位修饰,通过增大其分子量,降低肾脏消除率,提高药物体内半衰期;偶联PEG的立体屏蔽效应,减缓了胰蛋白酶的降解,同时也使其免疫原性大大降低;由于PEG的生物相容性好、药物半衰期的延长,可以减少用药次数,也相应的降低了其毒副作用。
本课题具体研究内容如下:
一、通过对PEG修饰反应的主要影响因素摩尔比、反应温度、反应时间、反应pH值的筛选,建立了一种优化的PEG-L-ASP的制备及纯化工艺,其优化的修饰条件为L-ASP:mPEG=1:10,温度37℃,时间6h,pH=5.0,酶溶液浓度为5.0mg/ml,该优化条件下L-ASP的修饰转化率为87.94%;建立了离子交换色谱和凝胶过滤色谱联用的分离纯化工艺,并用其分离纯化了优化修饰条件下制备的PEG-L-ASP,得到了修饰产物均一、且纯度在95%以上的修饰酶。
二、为了考察修饰产物PEG-L-ASP的理化性质及确证其一级结构,采用紫外扫描、IEF电泳、荧光胺法测修饰度、Western Blot免疫印迹、肽图分析等方法对偶联终产物PEG-L-ASP进行了分析。结果发现,纯化后的PEG-L-ASP的效价为134.076单位/mg,而L-ASP原酶的效价为373.670单位/mg,其修饰酶的活性保留为原酶的35.88%,修饰酶PEG-L-ASP与L-ASP原酶的最大吸收峰一样都在278nm的位置,等电点也都位于4.65～4.75之间,获得修饰酶PEG-L-ASP的PEG化率为3.27%,PEG-L-ASP修饰带与L-ASP未修饰带在Western Blot免疫印迹时都呈阳性反应,但PEG-L-ASP修饰带与抗体结合能力很弱,初步说明其免疫原性低,胰
The L-asparaginase (L-ASP) is one kind of white or light yellow freeze-dry dust, smell-less and tasteless. It is composed of homeotype tetramer and its relative molecular weight is 133000±5000. The main function is to hydrolyze aspartamic acid in blood serum to aspartic acid and the ammonia. L-ASP in clinical is mainly used to treat the acute lymphocyte leukemia (abbreviation AL). And the aspartamic acid is a must for cell protein synthetic and proliferation. Normal cells can synthesize aspartamic acid, but tumor cells of acute leukemia etc do not have that function. Thus when L-ASP causes the aspartamic acid sudden missing, the tumor cell cannot obtain enough aspartamic acid from the blood, and cannot synthesize either. Then massive tumor cells will be destructed because the protein synthesis is blocked and the proliferation is suppressed. L-ASP can also disturb the process of DNA and RNA synthesis in cells, possibly by affecting the G1 multiplication cycle of cells. In clinical practice, reorganized L-ASP also has short half-life, strong immunogenicity and poisonous side effect as other reorganized products. Because its half-life is short, it must be injected every day to maintain effective blood concentration. As a result, long-term and frequent injection increases the treatment cost, brings serious pain and inconvenience to patients, reduces the compliance and aggravate patients’economical burden.
In order to overcome the clinical limit of L-ASP, this research chose the single methoxy polyethylene glycol propylaldehyde (mPEG-ALD) to carry out localized modification on the N-terminal of the laevo-rotatory asparaginase. By increasing its molecular weight, the kidney elimination rate was reduced and in vivo half-life was enhanced; Because of stereo barrier effect of coupling PEG three-dimensional, the trypsin degradation was slowed down and its immunogenicity was also greatly reduced simultaneously; and because of the PEG biological good compatibility and lengthening of half-life, the injection amount of L-ASP was reduced, which correspondingly reduced its poisonous side effect.
The detailed contents of research were described as the following:
1 This research established one optimized PEG-L-ASP preparation system by screening proper mole ratio, reaction temperature, reaction time, pH value of the modification reaction of PEG. The optimized preparation condition was: mPEG was 1:10;temperature was 37 centigrade; time was 6 hours; pH value was 5; enzyme solution
为了克服L-ASP在临床用药上的限制,本课题选择单甲氧基聚乙二醇丙醛(mPEG-ALD)对左旋门冬酰胺酶(L-ASP)进行N-末端定位修饰,通过增大其分子量,降低肾脏消除率,提高药物体内半衰期;偶联PEG的立体屏蔽效应,减缓了胰蛋白酶的降解,同时也使其免疫原性大大降低;由于PEG的生物相容性好、药物半衰期的延长,可以减少用药次数,也相应的降低了其毒副作用。
本课题具体研究内容如下:
一、通过对PEG修饰反应的主要影响因素摩尔比、反应温度、反应时间、反应pH值的筛选,建立了一种优化的PEG-L-ASP的制备及纯化工艺,其优化的修饰条件为L-ASP:mPEG=1:10,温度37℃,时间6h,pH=5.0,酶溶液浓度为5.0mg/ml,该优化条件下L-ASP的修饰转化率为87.94%;建立了离子交换色谱和凝胶过滤色谱联用的分离纯化工艺,并用其分离纯化了优化修饰条件下制备的PEG-L-ASP,得到了修饰产物均一、且纯度在95%以上的修饰酶。
二、为了考察修饰产物PEG-L-ASP的理化性质及确证其一级结构,采用紫外扫描、IEF电泳、荧光胺法测修饰度、Western Blot免疫印迹、肽图分析等方法对偶联终产物PEG-L-ASP进行了分析。结果发现,纯化后的PEG-L-ASP的效价为134.076单位/mg,而L-ASP原酶的效价为373.670单位/mg,其修饰酶的活性保留为原酶的35.88%,修饰酶PEG-L-ASP与L-ASP原酶的最大吸收峰一样都在278nm的位置,等电点也都位于4.65～4.75之间,获得修饰酶PEG-L-ASP的PEG化率为3.27%,PEG-L-ASP修饰带与L-ASP未修饰带在Western Blot免疫印迹时都呈阳性反应,但PEG-L-ASP修饰带与抗体结合能力很弱,初步说明其免疫原性低,胰
The L-asparaginase (L-ASP) is one kind of white or light yellow freeze-dry dust, smell-less and tasteless. It is composed of homeotype tetramer and its relative molecular weight is 133000±5000. The main function is to hydrolyze aspartamic acid in blood serum to aspartic acid and the ammonia. L-ASP in clinical is mainly used to treat the acute lymphocyte leukemia (abbreviation AL). And the aspartamic acid is a must for cell protein synthetic and proliferation. Normal cells can synthesize aspartamic acid, but tumor cells of acute leukemia etc do not have that function. Thus when L-ASP causes the aspartamic acid sudden missing, the tumor cell cannot obtain enough aspartamic acid from the blood, and cannot synthesize either. Then massive tumor cells will be destructed because the protein synthesis is blocked and the proliferation is suppressed. L-ASP can also disturb the process of DNA and RNA synthesis in cells, possibly by affecting the G1 multiplication cycle of cells. In clinical practice, reorganized L-ASP also has short half-life, strong immunogenicity and poisonous side effect as other reorganized products. Because its half-life is short, it must be injected every day to maintain effective blood concentration. As a result, long-term and frequent injection increases the treatment cost, brings serious pain and inconvenience to patients, reduces the compliance and aggravate patients’economical burden.
In order to overcome the clinical limit of L-ASP, this research chose the single methoxy polyethylene glycol propylaldehyde (mPEG-ALD) to carry out localized modification on the N-terminal of the laevo-rotatory asparaginase. By increasing its molecular weight, the kidney elimination rate was reduced and in vivo half-life was enhanced; Because of stereo barrier effect of coupling PEG three-dimensional, the trypsin degradation was slowed down and its immunogenicity was also greatly reduced simultaneously; and because of the PEG biological good compatibility and lengthening of half-life, the injection amount of L-ASP was reduced, which correspondingly reduced its poisonous side effect.
The detailed contents of research were described as the following:
1 This research established one optimized PEG-L-ASP preparation system by screening proper mole ratio, reaction temperature, reaction time, pH value of the modification reaction of PEG. The optimized preparation condition was: mPEG was 1:10;temperature was 37 centigrade; time was 6 hours; pH value was 5; enzyme solution
引文
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[2] 姜忠义,高蓉,许松伟,王艳强.药物蛋白的聚乙二醇修饰[J]. 中国药学杂志,2002,37(6):409-412.
[3] Sidney Shifrin, Barbara G. Solis. L-Asparaginase from Escherichia coli B Chemical Modification of tyrosyl Residues[J]. The Journal of Biological Chemistry, 1972, 13(247) : 4121-4125.
[4] Vaulont S, Cognet MV, Kahn A. Glucose Regulation of Gene Transcription[J]. J Biol Chem, 2000, 275(41): 31555-31558.
[5] Takagaki K, Katsuma S, Horio T. cDNA microarray analysis of altered gene expression in Ara-C-treated leukemia cells[J]. Bioch Biophys Res Commun, 2003, 309(5): 351-358.
[6] Zhong C, Chen C, Michael S. Characterization of nutrientsensing response unit in the human asparagines systhetase promoter[J]. Biochem J, 2003, 372(2):603-609.
[7] Krejci O, Starkova J, Otova B, et al. Upregulation of asparagines synthetase fails to avert cell cycle arrest induced by L-asparaginase in TEL/AML1-posisitive leukaemic cells[J]. Leukemia, 2004,18(3): 434-441.
[8] 王钦超. L-天冬胺酰酶Ⅱ的发酵及分离纯化工艺研究[M]. 华东理工大学,2005.
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