在大肠杆菌中使用聚蛋白策略有效地产生人类线粒体沙佩罗宁 (Hsp60/Hsp10)
Sreejith Raran-Kurussi1, Pragyan P Parida1, Akanksha Aphale1
1Tata Institute of Fundamental Research Hyderabad, 36/P Gopanpally Village, Ranga Reddy District, Serilingampally, Hyderabad, Telangana, 500046, India.
Protein expression and purification
|July 25, 2025
概括
这项研究提出了一种新的方法,用于在大肠杆菌中产生功能性人类线粒体辅导素 (Hsp60/Hsp10). 一种多蛋白质策略确保了等等子子单元的产生,克服了重组蛋白质表达的静态度失衡.
科学领域:
- 生物化学 生化学
- 分子生物学分子生物学
- 蛋白质工程是指蛋白质工程.
背景情况:
- 大肠杆菌是重组蛋白质生产的主要宿主.
- 大肠杆菌中多蛋白质复合体的共同表达往往导致静态度失衡,阻碍精确的亚单元组合.
- 在异质宿主中可靠的多蛋白质复合物的过度生产对于研究它们的结构和功能至关重要.
研究的目的:
- 开发一种方法,以在大肠杆菌中产生人类线粒体沙佩罗宁 (Hsp60/Hsp10) 复合物.
- 为了克服多蛋白质复合体生产中石化学失衡的挑战.
- 为了确保产生的沙佩罗宁复合物的功能完整性.
主要方法:
- 采用了聚蛋白策略,将Hsp60和Hsp10亚单元融合在一起.
- 烟草静脉斑点病毒 (TVMV) 蛋白酶用于体内分离聚蛋白.
- 电视MV蛋白酶和Hsp60/Hsp10多蛋白是从同一个E. coli中的等离子体中共同表达的.
主要成果:
- 成功地产生了相当数量的成熟的Hsp60和Hsp10子单位.
- 获得了足够的TVMV蛋白酶活性,使多蛋白质完全分裂.
- 再组合的人类线粒体Chaperonin复合体被证明是完全功能性的.
结论:
- 描述的多蛋白质分裂策略有效地解决了多蛋白质复合体生产中的静态度失衡问题.
- 这种方法使得大肠杆菌中人类线粒体沙佩罗宁的可靠和功能性生产成为可能.
- 这种方法对其他具有挑战性的多蛋白质复合体的异质生产具有前景.
相关概念视频
Molecular Chaperones and Protein Folding
18.5K
The native conformation of a protein is formed by interactions between the side chains of its constituent amino acids. When the amino acids cannot form these interactions, the protein cannot fold by itself and needs chaperones. Notably, chaperones do not relay any additional information required for the folding of polypeptides; the native conformation of a protein is determined solely by its amino acid sequence. Chaperones catalyze protein folding without being a part of the folded protein.
The...
The...
18.5K
Mitochondrial Protein Sorting
4.4K
Mitochondria are double-membrane organelles of the eukaryotes involved in cellular metabolism, signaling, ATP synthesis, and programmed cell death. Each of these processes requires specific proteins and enzymes that must be correctly sorted to the right mitochondrial subcompartment for the proper functioning of the organelle.
Most of these mitochondrial proteins are encoded by the nucleus and imported to the mitochondria as unfolded or loosely folded precursors. Mitochondrial precursors...
Most of these mitochondrial proteins are encoded by the nucleus and imported to the mitochondria as unfolded or loosely folded precursors. Mitochondrial precursors...
4.4K
Bacterial Protein Maturation
88
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...
88
Mitochondrial Precursor Proteins
2.6K
Mitochondrial precursors are partially unfolded or loosely folded polypeptide chains. Newly synthesized precursors are inhibited from spontaneously folding into their native conformation by the cytosolic chaperones, heat shock proteins 70 (Hsp70), and mitochondrial import stimulation factors (MSFs). Precursors bound to MSFs are guided to the TOM70-TOM37 receptors, while precursors bound to Hsp70 chaperones are targetted to TOM20-TOM22 receptor complexes.
Most of the mitochondrial...
Most of the mitochondrial...
2.6K
Energy to Drive Translocation
2.1K
Mitochondrial protein import is powered by two distinct energy sources: ATP hydrolysis and electrochemical potential across the inner membrane. Newly synthesized precursors are bound by cytosolic chaperones of the Hsp70 family, which guide them to the import receptors on the mitochondrial surface. Utilizing the energy of ATP hydrolysis, Hsp70 chaperones transfer these precursors to the TOM receptors on the mitochondrial outer membrane.
Generally, polypeptides are unfolded by two distinct...
Generally, polypeptides are unfolded by two distinct...
2.1K
Porin Insertion in the Outer Mitochondrial Membrane
3.3K
Porins are beta-barrel proteins translocated to the mitochondrial outer membrane through the TOM complex into the intermembrane space. Porin precursors bind TIM chaperones within the intermembrane space and are guided to the Sorting and Assembly Machinery complex or SAM complex on the outer mitochondrial membrane.
Three models describe the assembly of porins by the SAM complex and their insertion into the outer membrane. Model 1 suggests that porins are assembled outside the SAM channel as the...
Three models describe the assembly of porins by the SAM complex and their insertion into the outer membrane. Model 1 suggests that porins are assembled outside the SAM channel as the...
3.3K


