重组人载脂蛋白C-I在毕赤酵母中的分泌表达及鉴定

中国生物工程杂志

2012, Vol. 32

Issue (08): 9-13

研究报告

重组人载脂蛋白C-I在毕赤酵母中的分泌表达及鉴定

苏曼曼¹, 陈月¹, 王丁丁², 许天敏³, 颜炜群¹

1. 吉林大学药学院长春 130021;
2. 广东药学院生命科学与生物制药学院广州 510006;
3. 吉林大学第二医院长春 130041

Expression and Identification of Recombinant Human Apolipoprotein C-I in Pichia pastoris

SU Man-man¹, CHEN Yue¹, WANG Ding-ding², XU Tian-min³, YAN Wei-qun ¹

1. College of Pharmacy, Jilin University, Changchun 130021,China;
2. College of Life Science and Biopharmacology, Guangdong Pharmaceutical University, Guangzhou 510006,China;
3. The Second Hospital, Jilin University, Changchun 130041, China

全文: PDF(519 KB) HTML

摘要： 目的:在毕赤酵母中高效分泌表达与天然人载脂蛋白C-I具有相同结构和活性的重组人载脂蛋白C-I(rhApoC-I)。方法:RT-PCR法自人肝组织调取编码人ApoC-I的cDNA,构建真核分泌型表达载体pPICZα/hApoC-I。重组质粒线性化后转化毕赤酵母感受态细胞,甲醇诱导表达,建立rhApoC-I的毕赤酵母表达体系。对rhApoC-I进行Western blot分析和体外活性研究。结果:经PCR法克隆的hApoC-I cDNA序列与GenBank登录序列一致。SDS-PAGE和Western blot分析均在分子量约6.6kDa出现特异性条带,2L发酵条件下表达量达到80mg/L。结论:首次在毕赤酵母菌中高效分泌表达rhApoC-I,并确定其具有抑制血小板衍生生长因子诱导的平滑肌细胞增殖的抑制作用,为进一步研究其结构与功能提供物质基础。

关键词： 载脂蛋白C-I; 分泌表达; 毕赤酵母; 平滑肌细胞

Abstract: Apolipoprotein C-I (ApoC-I) is a small, basic apolipoprotein which is mainly secreted by the liver as a component of triglyceride-rich lipoproteins and high density lipoproteins whose importance in plasma lipoprotein metabolism is increasingly evident. Therefore, a Pichia pastoris expression system was first used to obtain a high level expression of secreted, recombinant human ApoC-I (rhApoC-I). The full-length ApoC-I encoding sequence, gained by RT-PCR, was inserted into the pPICZαC vector and transform Pichia pastoris strain X33 after sequencing. High expression transformants with zeocin resistance were detected by SDS-PAGE and Western blot analysis. The antiproliferation effects of rhApoC-I on rat coronary artery smooth muscle cells was analysed by the methylthiazoletetrazolium (MTT) assay. It was showed that there were rhApoC-I(6.6kDa) in the culture supernatant induced by methanol. The expression level of rhApoC-I was 80mg/L. RhApoC-I can inhibit the proliferation which induced by PDGF remarkably. Ground on this, the composition and functions of rhApoC-I can be studied.

Key words: Apolipoprotein C-I Secretory expression Pichia pastoris Smooth muscle cells

收稿日期: 2012-04-19 出版日期: 2012-08-25

ZTFLH:

Q786

基金资助: 吉林省发展与改革委员会产业技术研究与开发专项(20100016)资助项目

通讯作者: 许天敏, 颜炜群 E-mail: tianminxu@yahoo.cn;weiqun.yan@yahoo.com.cn

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	苏曼曼
	陈月
	王丁丁
	许天敏
	颜炜群

引用本文:

苏曼曼, 陈月, 王丁丁, 许天敏, 颜炜群. 重组人载脂蛋白C-I在毕赤酵母中的分泌表达及鉴定[J]. 中国生物工程杂志, 2012, 32(08): 9-13.

SU Man-man, CHEN Yue, WANG Ding-ding, XU Tian-min, YAN Wei-qun. Expression and Identification of Recombinant Human Apolipoprotein C-I in Pichia pastoris. China Biotechnology, 2012, 32(08): 9-13.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2012/V32/I08/9

[1] Gautier T, Masson D, de Barros J P, et al. Human apolipoprotein C-I accounts for the ability of plasma high density lipoproteins to inhibit the cholesteryl ester transfer protein activity. J Biol Chem, 2000,275 (48):37504-37509.
[2] Xu Y, Berglund L, Ramakrishnan R, et al. A common Hpa I RFLP of apolipoprotein C-I increases gene transcription and exhibits an ethnically distinct pattern of linkage disequilibrium with the alleles of apolipoprotein E. J Lipid Res, 1999,40 (1): 50-58.
[3] Polz E, Kotite L, Havel R J, et al. Human apolipoprotein C-I:concentration in blood serum and lipoproteins. Biochem Med, 1980,24 (3): 229-237.
[4] Bucay N, Lauer S J, Taylor J M. A far-downstream hepatocyte-specific control region directs expression of the linked human apolipoprotein E and C-I genes in transgenic mice. J Biol Chem, 1993,268 (11): 8221-8229.
[5] Malmendier C L, Lontie J F, Grutman G A, et al. Metabolism of apolipoprotein C-I in normolipoproteinemic human subjects. Atherosclerosis, 1986,62 (2): 167-172.
[6] Kowal R C, Herz J, Weisgraber K H, et al.Opposing effects of apolipoproteins E and C on lipoprotein binding to low density lipoprotein receptor-related protein. J Biol Chem, 1990,265 (18): 10771-10779.
[7] Weisgraber K H, Mahley R W, Kowal R C, et al.Apolipoprotein C-I modulates the interaction of apolipoprotein E with beta-migrating very low density lipoproteins (beta-VLDL) and inhibits binding of beta-VLDL to low density lipoprotein receptor-related protein. J Biol Chem, 1990,265 (36): 22453-22459.
[8] Knott T J, Robertson M E, Priestley L M.Characterisation of mRNAs encoding the precursor for human apolipoprotein C I. Nucleic Acids Res, 1984,12 (9): 3909-3915.
[9] Jonas A, Sweeny S A, Herbert P N. Discoidal complexes of A and C apolipoproteins with lipids and their reactions with lecithin: cholesterol acyltransferase. J Biol Chem, 1984,259 (10): 6369-6375.
[10] Steyrer E, Kostner G M. Activation of lecithin-cholesterol acyltransferase by apolipoprotein D: comparison of proteoliposomes containing apolipoprotein D, A-I or C-I. Biochim Biophys Acta, 1988,958 (3): 484-491.
[11] James P F, Dogovski C, Dobson R C, et al. Aromatic residues in the C-terminal helix of human apoC-I mediate phospholipid interactions and particle morphology. J Lipid Res, 2009,50(7):1384-1394
[12] Su M,Bao X,Wang W,et al.Expression and identification of recombinant human apolipoprotein E3 in Pichia pastoris.Chinese Journal of Gerontology,2009,29(7):841-843.
[13] Wang D,Su M,Hu L,et al. A vector system for the production of single-chain Fv-Fc fusions in Pichia pastoris. China Biotechnology,2011,31(8):110-117.
[14] Gurkan C, Ellar D J. Recombinant production of bacterial toxins and their derivatives in the methylotrophic yeast Pichia pastoris. Microb Cell Fact, 2005,4: 33.
[15] Kolmakova A, Kwiterovich P, Virgil D, et al. Apolipoprotein C-I induces apoptosis in human aortic smooth muscle cells via recruiting neutral sphingomyelinase. Arterioscler Thromb Vasc Biol, 2004,24 (2):264-269.

[1]	陈中伟,郑璞,陈鹏程,吴丹. 耐热植酸酶突变体的筛选及性质研究 ^*[J]. 中国生物工程杂志, 2021, 41(2/3): 30-37.
[2]	陈鑫洁,钱芷兰,刘启,赵清,张元兴,蔡孟浩. 毕赤酵母底盘芳香族氨基酸合成途径改造生产肉桂酸及对香豆酸^*[J]. 中国生物工程杂志, 2021, 41(10): 52-61.
[3]	石鹏程, 纪晓俊. 酵母系统表达人表皮生长因子研究进展 ^*[J]. 中国生物工程杂志, 2021, 41(1): 72-79.
[4]	章小毛,郭敬涵,洪解放,陆海燕,丁娟娟,邹少兰,范寰. **UPRE-lac Z为报告基因评价酵母UPR响应初步研究 ^***[J]. 中国生物工程杂志, 2020, 40(10): 1-9.
[5]	胡艳红,龚雪梅,丁柳柳,高嵩,李婷婷. 利用短短芽孢杆菌进行酮还原酶CgKR2的高效表达与纯化 ^*[J]. 中国生物工程杂志, 2019, 39(8): 59-65.
[6]	田园,李艳玲. 基于重组毕赤酵母的fusaruside生物合成 ^*[J]. 中国生物工程杂志, 2019, 39(7): 8-14.
[7]	彭强强,刘启,徐名强,张元兴,蔡孟浩. 新型重组毕赤酵母产人胰岛素前体的表达工艺研究 ^*[J]. 中国生物工程杂志, 2019, 39(7): 48-55.
[8]	严建,贾禄强,丁健,史仲平. 甲醇周期诱导控制强化毕赤酵母生产猪α干扰素 ^*[J]. 中国生物工程杂志, 2019, 39(6): 32-40.
[9]	姚银,闵琪,熊海容,张莉. 木聚糖酶和甘露聚糖酶在毕赤酵母中的共表达及产酶分析 ^*[J]. 中国生物工程杂志, 2019, 39(3): 37-45.
[10]	张文玉,魏东升,钱江潮. 共表达PDI1、MDH1和HAC1基因对重组毕赤酵母分泌表达葡糖氧化酶的影响 ^*[J]. 中国生物工程杂志, 2019, 39(10): 24-33.
[11]	王彤,徐岩,喻晓蔚. 毕赤酵母Kex2蛋白酶的同源表达及酶学性质 ^*[J]. 中国生物工程杂志, 2019, 39(1): 38-45.
[12]	程功,焦思明,任立世,冯翠,杜昱光. 枯草芽孢杆菌壳聚糖酶水解制备低脱乙酰度壳寡糖及其组分分析 ^*[J]. 中国生物工程杂志, 2018, 38(9): 19-26.
[13]	唐健雪,肖永乐,彭俊杰,赵世纪,万小平,高荣. 融合抗菌肽基因在重组毕赤酵母的表达及体外活性研究 ^*[J]. 中国生物工程杂志, 2018, 38(6): 9-16.
[14]	张潘潘,许延吉,王之可,刘晓,李素霞. 重组猪胰蛋白酶及其R122位点突变体在毕赤酵母中的高效表达及其性质研究[J]. 中国生物工程杂志, 2018, 38(5): 56-65.
[15]	黄鹏,杜望春,施尉珺,饶玉良,孙庆文,张宁. 人源类溶菌酶蛋白6的功能研究及生理特性分析 ^*[J]. 中国生物工程杂志, 2018, 38(3): 1-8.

Viewed

Full text

Abstract

Cited

Shared

Discussed