通过改进的核糖体,增强D-氨基酸的融入蛋白质
Larisa M Dedkova1, Nour Eddine Fahmi, Serguei Y Golovine
1Departments of Chemistry and Biology, University of Virginia, Charlottesville, VA 22904, USA.
Journal of the American Chemical Society
|May 29, 2003
概括
经过修改的大肠杆菌核糖体使得D-氨基酸能够更好地被纳入蛋白质中. 一些由此产生的蛋白质,如二叶酸还原酶,保留了充分的功能,证明了新型蛋白质工程的潜力.
科学领域:
- 分子生物学分子生物学
- 生物化学 生物化学
- 蛋白质工程是指蛋白质工程.
背景情况:
- 核糖体对于蛋白质合成至关重要,将遗传密码转化为功能性蛋白质.
- 23S核糖体RNA (rRNA) 是大型核糖体亚单元的关键组成部分,参与基转移酶活性.
- 将非正规氨基酸纳入蛋白质可以扩大它们的功能能力.
研究的目的:
- 研究23SrRNA特定区域突变对核糖体功能的影响.
- 为了确定修改后的核糖体是否可以将D-氨基酸纳入蛋白质.
- 评估D-氨基酸融入特定蛋白质的功能后果.
主要方法:
- 从多重复制等离子体中过度表达改造的Escherichia coli 23S rRNA,从而产生突变的核糖体.
- 突变发生和选择以识别23S rRNA基转移酶中心 (2447-2450) 和螺旋89 (2457-2462) 中突变的克隆.
- 使用这些突变的核糖体制备无细胞蛋白质合成系统,并分析D-氨基酸融入二叶酸减少酶 (DHFR) 和火虫光酶的分析.
主要成果:
- 在23SrRNA区域2447-2450和2457-2462的突变被成功引入和表征.
- 从这些突变核糖体中获得的无细胞系统显示,D-氨酸和D-氨酸在标蛋白中被显著增强.
- 在大肠杆菌DHFR和Photinus pyralis火 luciferase的特定位置发生了D-氨基酸的结合.
- 一些修改后的蛋白质,特别是D-氨基酸替代在位置10的DHFR类似物,保留了与它们的L-氨基酸对应物相比的完全酶活性.
- 在其他位置的替代导致蛋白质的活性显著降低.
结论:
- 特定23S rRNA区域的突变可以产生能够将D-氨基酸纳入蛋白质中的核糖体.
- 含有D-氨基酸的蛋白质的功能完整性取决于位置.
- 这项研究展示了使用非正规氨基酸设计蛋白质的新方法,可能导致新的治疗或工业应用.
相关概念视频
Protein Glycosylation
Glycosylation, the most common post-translational modification for proteins, serves diverse functions. Adding sugars to proteins makes the proteins more resistant to proteolytic digestion. Glycosylated proteins can act as markers and receptors to promote cell-cell adhesion. Additionally, they have many essential quality control functions in the cell, such as correct protein folding and facilitating transport of misfolded proteins to the cytosol, which can be degraded.
Glycosylation occurs in...
Glycosylation occurs in...
Oligosaccharide Assembly
Protein glycosylation starts in the ER lumen and continues in the Golgi apparatus. Glycosyltransferases catalyze the addition of sugar molecules or glycosylation of proteins. Usually, these enzymes add sugars to the hydroxyl groups of selected serine or threonine residues to form O-linked glycans or the amino groups of asparagine residues to form N-linked glycans. Different positions on the same polypeptide chain can contain differently linked glycans.
Multiple sugar molecules that may or may...
Multiple sugar molecules that may or may...
Protein Folding Quality Check in the RER
ER is the primary site for the maturation and folding of soluble and transmembrane secretory proteins. The calnexin cycle is a specific chaperone system that folds and assesses the confirmation of N-glycosylated proteins before they can exit the ER lumen. The primary players of this quality check pipeline are the lectins, ER-resident chaperones, and a glucosyl transferase enzyme. In case the calnexin system in the lumen fails to salvage a misfolded protein, it is transported to the cytoplasm...
Insulin Secretory Vesicles
Insulin secretory vesicles release insulin to stimulate blood glucose uptake and regulate carbohydrate metabolism. When the blood glucose levels increase, glucose enters the pancreatic β-islet cells through glucose transporters. Once inside, glucose is metabolized through glycolysis, the citric acid cycle, and the electron transport chain, producing ATP. This increase in ATP concentration closes ATP-sensitive potassium channels, leading to depolarization of the membrane and the opening of...
Bacterial Protein Maturation
Bacterial protein maturation is a tightly regulated process that ensures newly synthesized polypeptides achieve correct functional conformations. This maturation involves a series of modifications, folding events, and quality control steps, often assisted by specialized chaperone proteins.N-Terminal ModificationsThe maturation of bacterial polypeptides begins cotranslationally as the polypeptide exits the ribosome. The first amino acid, N-formylmethionine (fMet), is typically modified at the...
Production of Pharmaceuticals
Industrial insulin production uses genetically engineered E. coli expressing a proinsulin gene controlled by a tryptophan promoter and containing a methionine linker for later cleavage. The cells also carry ampicillin resistance for selective growth. Seed cultures are stored at −80 °C and production begins by thawing a small amount to inoculate starter cultures, which are progressively scaled to a 50,000-L bioreactor. In the bioreactor, E. coli grow in nutrient-rich media under sterile, tightly...


