靶向肽位置影响融合蛋白与细胞结合活性

中国生物工程杂志

2012, Vol. 32

Issue (6): 74-78

技术与方法

靶向肽位置影响融合蛋白与细胞结合活性

王海, 石必枝, 杨胜利, 李宗海, 罗晓莹

上海交通大学医学院附属仁济医院上海市肿瘤研究所癌基因及相关基因国家重点实验室上海 200032

Location of EGFP Tag Affects the Binding Activity of Peptide Fusions

WANG Hai, SHI Bi-zhi, YANG Sheng-li, LI Zong-hai, LUO Xiao-ying

State Key Laboratory of Oncogenes and Related Genes, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai Cancer Institute, Shanghai 200032, China

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摘要： 目的:探讨靶向多肽与标签蛋白(绿色荧光蛋白)的位置关系是否会影响融合蛋白与细胞之间的结合能力。方法:将获得的GE11和LyP1两种靶向多肽分别与增强型绿色荧光蛋白在不同位置融合表达,通过原核系统表达纯化,将纯化的蛋白加入血清饥饿的SMMC-7721肝癌细胞株培养液中,处理3h,通过荧光显微镜观察细胞中绿色荧光蛋白的情况检查融合多肽与细胞的结合情况。结果:绿色荧光蛋白的羧基端和GE11、LyP1多肽分别融合表达,处理细胞后,融合蛋白显示与细胞有很强的结合能力;当GE11、LyP1在绿色荧光蛋白氨基端融合时,融合蛋白几乎不能与细胞结合。在此基础上,检测了多种靶向肽对多种细胞的靶向效应。结论:不合适的融合策略会降低,甚至消除靶向多肽的结合能力;融合大分子量蛋白也会改变靶向肽的靶向效应。因此,当使用靶向多肽携带基因进行研究时,其在融合蛋白中的位置应该非常谨慎。

关键词： 靶向肽; GE11; LyP1; RGD4c

Abstract: Purpose: To investigate whether the location between targeting peptide and tag protein (eGFP) would influence the binding capacity of fusing protein to cell. Methods: To get Ge11 and LyP1 fusing eGFP recombination protein by expressing system in E coli. respectively, and then put the recombination protein into the medium of serum starving SMMC-7721 liver cancer cell line, after treating 3 hours, to detect the binding capacity by Fluorescence microscopy. Results: when eGFP at the Carboxyl terminal of targeting peptides, the recombination protein could bind to the cell; But when eGFP at the Nitro terminal, the recombination protein loss the binding capacity. On the basis of these researches, the other 3 targeting peptides were detected. Conclusion: These results indicate that an inappropriate fusion strategy would lead to decreased or even totally loss of function of targeting peptides. Thus, it should be cautious when used EGFP as a tracer to study the targeting activities of peptides.

Key words: Targeting peptide GE11 LyP1 RGD4c

收稿日期: 2012-02-15 出版日期: 2012-06-25

ZTFLH:

Q819

基金资助: "十一五计划"(2008ZX10002-023)、国家高技术研究发展计划(2007AA021203)资助项目

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引用本文:

王海, 石必枝, 杨胜利, 李宗海, 罗晓莹. 靶向肽位置影响融合蛋白与细胞结合活性[J]. 中国生物工程杂志, 2012, 32(6): 74-78.

WANG Hai, SHI Bi-zhi, YANG Sheng-li, LI Zong-hai, LUO Xiao-ying. Location of EGFP Tag Affects the Binding Activity of Peptide Fusions. China Biotechnology, 2012, 32(6): 74-78.

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https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2012/V32/I6/74

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