hBmp4前导肽与成熟肽在毕赤酵母中的融合表达及其对成熟肽折叠的影响
发布时间：2018-07-14 12:45
【摘要】：骨形态发生蛋白(Bone Morphogenesis Proteins,BMPs)是一类具有诱 骨活性的蛋白质生长因子。骨形态发生蛋白.4(BMP4)是BMPs中诱导 成骨活性较强的一种。目前已有研究者尝试在不同的表达系统中进行重组 表达,但是在表达产物中,无法获得活性较高的二聚体形式的蛋白。BMP4 在胞内是以前体的形式合成的,包括信号肽、前导肽和成熟肽三个部分。 研究表明,在目前发现的绝大部分的以含前导肽的前体形式合成的蛋白质 在其前导肽不存在的情况下均无法正确折叠。因此认为,Pro肽在某些蛋 白质的折叠过程中扮演了分子内分子伴侣(IMC)的角色。本研究希望在 毕赤酵母中将BMP4前导肽与成熟肽融合表达,以此来探讨BMP4前导肽 在辅助成熟肽的折叠以及二硫键的配对过程中是否起到一定的作用。主要 的研究工作和研究结果包括:(1)将hBMP4前导肽与成熟肽片段克隆到 分泌型表达载体质粒pPIC9K多克隆位点上,构建能表达hBMP4前导肽 与成熟肽的重组质粒pPIC9K-hBMP4/pro-ori;(2)以这一表达载体为基础, 对hBMP4前导肽与成熟肽之间内源性蛋白水解酶识别位点进行定点突 变,将真核细胞中碱性氨基酸蛋白酶(furin)识别的位点(RXXR)改为酵 母细胞中丝氨酸蛋白酶(Kex2P)识别的位点Kex2,并以此构建了重组质 粒pPIC9K-hBMP4/pro-mut;(3)表达质粒经转化酵母宿主菌株GSll5, PCR鉴定基因整合后,摇瓶培养,甲醇诱导表达并用点杂交来快速筛选高 表达菌株。表达产物经SDS-PAGE和Western blotting分析表明表达产物 具有良好的抗原性和特异性,内源性蛋白水解酶识别位点的改造能够使前 导肽有效切割,切割后BMP4成熟肽的分子量为22KD,与标准蛋白一致, 但不存在同二聚体形式的产物。
[Abstract]:Bone Morphogenesis proteins (BMPs) are a kind of protein growth factors with bone inductive activity. Bone morphogenetic protein 4 (BMP4) is one of BMPs with high osteogenic activity. So far, researchers have tried to recombine in different expression systems, but in the expressed products, The protein. BMP4 in the form of high activity dimer can not be synthesized in the intracellular form, which consists of three parts: signal peptide, precursor peptide and mature peptide. It has been found that most of the proteins synthesized in the form of precursors containing precursor peptides can not be folded correctly without the existence of the precursor peptides. Therefore, it is suggested that protopeptide plays the role of intramolecular chaperone (IMC) in the folding of some egg white matter. In this study, we hope to express BMP4 prepeptide and mature peptide in Pichia pastoris. In order to investigate whether BMP4 prepeptide plays a role in assisting the folding of mature peptides and the matching of disulfide bonds. The main research work and results are as follows: (1) cloning hBMP4 precursor peptide and mature peptide fragment into secretory expression Vector plasmid pPIC9K polyclonal site, The recombinant plasmid pPIC9K-hBMP4 / pro-orii was constructed to express hBMP4 prepeptide and mature peptide. (2) based on this expression vector, hBMP4 prepeptide was synthesized. The identification site of endogenous proteolytic enzyme was suddenly changed with mature peptide. The (furin) recognition site (RXXR) of alkaline amino acid protease in eukaryotic cells was replaced by Kex2 site recognized by serine protease (Kex2P) in yeast cells, and the recombinant plasmid was constructed. PPIC9K-hBMP4 / pro-mutt; (3) the expression plasmid was identified after gene integration by the transformed yeast host strain GSll5. In shaking flask culture, methanol induced expression and dot hybridization were used to quickly screen high expression strains. SDS-PAGE and Western blotting analysis showed that the expressed product had good antigenicity and specificity. The modification of the recognition site of endogenous proteolytic enzyme can make the preconductors cut effectively. The molecular weight of the BMP4 mature peptide was 22kD, which was consistent with the standard protein, but there was no dimer product.
【学位授予单位】：福建师范大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：Q78;Q516
本文编号：2121690
[Abstract]:Bone Morphogenesis proteins (BMPs) are a kind of protein growth factors with bone inductive activity. Bone morphogenetic protein 4 (BMP4) is one of BMPs with high osteogenic activity. So far, researchers have tried to recombine in different expression systems, but in the expressed products, The protein. BMP4 in the form of high activity dimer can not be synthesized in the intracellular form, which consists of three parts: signal peptide, precursor peptide and mature peptide. It has been found that most of the proteins synthesized in the form of precursors containing precursor peptides can not be folded correctly without the existence of the precursor peptides. Therefore, it is suggested that protopeptide plays the role of intramolecular chaperone (IMC) in the folding of some egg white matter. In this study, we hope to express BMP4 prepeptide and mature peptide in Pichia pastoris. In order to investigate whether BMP4 prepeptide plays a role in assisting the folding of mature peptides and the matching of disulfide bonds. The main research work and results are as follows: (1) cloning hBMP4 precursor peptide and mature peptide fragment into secretory expression Vector plasmid pPIC9K polyclonal site, The recombinant plasmid pPIC9K-hBMP4 / pro-orii was constructed to express hBMP4 prepeptide and mature peptide. (2) based on this expression vector, hBMP4 prepeptide was synthesized. The identification site of endogenous proteolytic enzyme was suddenly changed with mature peptide. The (furin) recognition site (RXXR) of alkaline amino acid protease in eukaryotic cells was replaced by Kex2 site recognized by serine protease (Kex2P) in yeast cells, and the recombinant plasmid was constructed. PPIC9K-hBMP4 / pro-mutt; (3) the expression plasmid was identified after gene integration by the transformed yeast host strain GSll5. In shaking flask culture, methanol induced expression and dot hybridization were used to quickly screen high expression strains. SDS-PAGE and Western blotting analysis showed that the expressed product had good antigenicity and specificity. The modification of the recognition site of endogenous proteolytic enzyme can make the preconductors cut effectively. The molecular weight of the BMP4 mature peptide was 22kD, which was consistent with the standard protein, but there was no dimer product.
【学位授予单位】：福建师范大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：Q78;Q516
【参考文献】
相关期刊论文 前8条
1 王洪海,高卜渝,陈佩丽,董灵,李育阳;酵母表达基因工程产物不均一性分析及其对策[J];中国科学(B辑 化学 生命科学 地学);1994年12期
2 姚斌,张春义,王建华,范云六;高效表达具有生物学活性的植酸酶的毕赤酵母[J];中国科学C辑:生命科学;1998年03期
3 欧阳立明,张惠展,张嗣良,刘志敏;巴斯德毕赤酵母的基因表达系统研究进展[J];生物化学与生物物理进展;2000年02期
4 赵翔,霍克克,李育阳;毕赤酵母的密码子用法分析[J];生物工程学报;2000年03期
5 蔡传奇,方荣祥;毕赤酵母基因操作技术的改进及其在水蛭素表达中的应用[J];生物工程学报;2001年02期
6 赵明，，王会信，周廷冲;重组人骨形态发生蛋白－2成熟肽在大肠杆菌中的表达及其诱导成骨活性[J];生物化学杂志;1994年03期
7 隋少飞,陈松林;巴氏毕赤酵母表达系统的特点及其研究进展[J];生物技术通报;2004年03期
8 刘礼初,梁国栋,杜靖远,付士红,陈飞,郑启新,肖宝筠,侯云德;重组人骨形态发生蛋白-2/7融合基因的构建及其在大肠杆菌中的表达和初步活性测定[J];同济医科大学学报;1999年04期
本文编号：2121690
本文链接：https://www.wllwen.com/yixuelunwen/binglixuelunwen/2121690.html
最近更新
教材专著
