Investigation on the origin of charge heterogeneity of antibody by genetic engineering
基因工程研究抗体电荷异质性的起源
基本信息
- 批准号:12680618
- 负责人:
- 金额:$ 1.41万
- 依托单位:
- 依托单位国家:日本
- 项目类别:Grant-in-Aid for Scientific Research (C)
- 财政年份:2000
- 资助国家:日本
- 起止时间:2000 至 2001
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
1 Charge heterogeneity of recombinant Fab' should partially represent charge heterogeneity of immunoglobulin molecule. Deamidation of an asparagine residue adjacent to glycine and serine at its carboxyl side has been reported to be accelerated. Five asparagine residues of recombinant Fab' (mouse γl, K) and one glutamine residue were mutated to serine residue to produce six different mutants.2 The six recombinant Fab' and the original wild type Fab' were produced from E. coli and the Fab's were labeled at a cysteine residue in the hinge region with tetramethyl rhodamine dye. The labeled Fab's were purified by isoelectric focusing in an agarose slab gel to 99 % homogeneity.3 The labeled homogeneous Fab's were incubated at pH 7.5 for 15 h and 30 h, and the heterogeneity of the products in terms of pi was analyzed by the use of a house made capillary isoelectric focusing instrument with a scanning laser-induced fluorescence detector. Incubation produced acidic pi isoforms with the decrease of the original peak. When the velocity of the reduction of the original peaks were compared to that of the wild type, two mutants among the six showed marked reduction in the decrease. The results showed Ansl35 of γl chain and Asnl57 of κ chain contribute 65 % and 30 % of the overall charge heterogeneity of Fab' molecule, respectively.4 A double mutant of γlN135S and κN157S was constructed and its velocity to be heterogeneous was determined to be as small as 8 % in comparison with the wild type.5 The discovery of the quantitative contribution of the two asparagine reside in the charge heterogeneity of antibody is important in protein chemistry and would be useful in the production of stable affinity probes and antibody medicines.
1重组Fab'的电荷异质性应部分代表免疫球蛋白分子的电荷异质性。据报道,在其羧基侧与甘氨酸和丝氨酸相邻的天冬酰胺残基的脱酰胺化被加速。将重组Fab'(小鼠γ 1,K)的5个天冬酰胺残基和1个谷氨酰胺残基突变为丝氨酸残基,得到6个不同的突变体。用四甲基罗丹明染料在铰链区的半胱氨酸残基处标记Fab。通过琼脂糖平板凝胶中的等电聚焦将标记的Fab纯化至99%结晶度。3将标记的均质Fab在pH 7.5下孵育15小时和30小时,并通过使用自制的毛细管等电聚焦仪器和扫描激光诱导荧光检测器分析产物的异质性(以pi计)。孵育产生酸性pi亚型,原始峰降低。当将原始峰的降低速度与野生型的降低速度进行比较时,六个突变体中的两个突变体显示出降低的显著降低。结果表明,γ 1链的Ansl35和κ链的Asnl57分别占Fab ′分子总电荷异质性的65%和30%,4构建了γ 1N135S和κN157S双突变体,其异质化速度小至8%。发现两种天冬酰胺在抗体电荷异质性中的定量贡献在蛋白质化学中是重要的,并且将用于生产稳定的抗体。亲和探针和抗体药物。
项目成果
期刊论文数量(8)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Kiyohito Shimura: "Fluorescence-Labeled Peptide pI Markers for Capillary Isoelectric Focusing"Anal.Chem.. 74(in press). (2002)
Kiyohito Shimura:“用于毛细管等电聚焦的荧光标记肽 pI 标记”Anal.Chem.. 74(印刷中)。
- DOI:
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- 影响因子:0
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Kiyohito Shimura: "Immunoassay of Serum alpha 1-Antitrypsin by Affinity-Probe Capillary Isoelectric Focusing using a Fluorescence-Labeled Recombinant Antibody Fragment"Electrophoresis. 23(in press). (2002)
Kiyohito Shimura:“使用荧光标记的重组抗体片段通过亲和探针毛细管等电聚焦对血清 α1-抗胰蛋白酶进行免疫测定”电泳。
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- 影响因子:0
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K.Shimura et al: "Synthetic Oligopeptides as Isoelectric Point Markers for Capillary Isoelectric Focusing with UV absorption detection"Electrophoresis. 21・3. 603-610 (2000)
K. Shimura 等人:“用于毛细管等电聚焦与紫外吸收检测的合成寡肽”电泳 603-610。
- DOI:
- 发表时间:
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- 影响因子:0
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Kiyohito Shimura: "Immunoassay of Seram α_1-Antitrypsin by Affinity-Probe Capillary Isoelectric Focusing using a Fluorescence-Labeled Recombinant Antibody"Electrophoresis. 23(in press). (2002)
Kiyohito Shimura:“使用荧光标记重组抗体通过亲和探针毛细管等电聚焦进行血清 α_1-抗胰蛋白酶的免疫测定”23(印刷中)。
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- 影响因子:0
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Kiyohito Shimura et al.: "Fluorescence-Labeled Peptide pI Markers for Capillary Isoelectric Focusing"Anal. Chem.. Vol. 74 (in press). (2002)
Kiyohito Shimura 等人:“用于毛细管等电聚焦的荧光标记肽 pI 标记”分析。
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- 影响因子:0
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