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相关概念视频

Tagging and Fusion Proteins01:24

Tagging and Fusion Proteins

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Proteins are involved in several cellular processes and biochemical reactions. Analyzing a specific protein of interest requires it to be isolated from the other proteins in the cell. This is achieved by overexpressing the specific gene in a suitable host to produce large quantities of the target protein. A tag or label is recombined with the gene to produce a fusion protein containing the target protein and the tag. The tags on these fusion proteins can then be used for easy detection and...
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Tail-anchoring of Proteins in the ER Membrane01:45

Tail-anchoring of Proteins in the ER Membrane

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Tail-anchored, or TA, proteins are estimated to make up to 3-5% of membrane proteins found in the eukaryotic cell. Such proteins have a single transmembrane domain located approximately 30 amino acid residues upstream from the C-terminal end. As a result, the signal recognition particle (SRP) cannot guide a TA protein to the ER membrane for cotranslational insertion. Hence, they are integrated into the ER membrane post-translationally using their C-terminal end as the anchor. TA proteins...
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From DNA to Protein03:06

From DNA to Protein

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The flow of genetic information in cells from DNA to mRNA to protein is described by the central dogma, which states that genes specify the sequence of mRNAs, which in turn specify the sequence of amino acids making up all proteins. The decoding of one molecule to another is performed by specific proteins and RNAs. Because the information stored in DNA is so central to cellular function, it makes intuitive sense that the cell would make mRNA copies of this information for protein synthesis...
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Leaky Scanning02:28

Leaky Scanning

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During most eukaryotic translation processes, the small 40S ribosome subunit scans an mRNA from its 5' end until it encounters the first start AUG codon. The large 60S ribosomal subunit then joins the smaller one to initiate protein synthesis. The location of the translation initiation is largely determined by the nucleotides near the start codon as there may be multiple translation initiation sites present on the mRNA.  Marilyn Kozak discovered that the sequence RCCAUGG (where R...
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Transfer RNA Synthesis02:36

Transfer RNA Synthesis

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One of the unique features of tRNA is the presence of modified bases. In some tRNAs, modified bases account for nearly 20% of the total bases in the molecule. Altogether, these unusual bases protect the tRNA from enzymatic degradation by RNases.
Each of these chemical modifications is carried by a specific enzyme, post-transcription. All of these enzymes have unique base and site-specificity. Methylation, the most common chemical modification, is carried by at least nine different enzymes, with...
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Translocation of Proteins into the Mitochondria01:19

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Mitochondrial precursors are translocated to the internal subcompartments via independent mechanisms involving distinct protein machineries called translocases.
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A Fast and Quantitative Method for Post-translational Modification and Variant Enabled Mapping of Peptides to Genomes
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在多链内插入的新型His-tag变体.

Anastasiia G Tarabarova1, Anton Lopukhov2, Alexey N Fedorov3

  • 1A N Bach Institute of Biochemistry of the Russian Academy of Sciences, Leninskii prosp 33/2, Moscow 119071, Russian Federation.

ACS omega
|January 15, 2024
PubMed
概括

新设计的内部His标签,具有histidine和glycine残留物,最大限度地减少蛋白质结构的影响. 这些可插入的标签提供了一种新的方法来净化各种蛋白质,而无需N或C端融合.

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Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System
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科学领域:

  • 生物化学 生物化学
  • 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:

  • 六西丁 (His-tag) 亲和度净化是一种标准的技术.
  • N-或C-终端的His-tag融合可以对蛋白质结构和功能产生负面影响.
  • 探索了内部标签插入,以减轻N / C终端融合问题.

研究的目的:

  • 设计和描述新的内部His标签.
  • 为了最大限度地减少固体障碍和对蛋白质结构/功能的影响.
  • 扩大像eGFP和GrAD207.7这样的融合蛋白的实用性.

主要方法:

  • 设计了内部的His标签,其中有两个由甘氨酸残留物分离的histidine三胞胎.
  • 通过将它们内部融合到增强绿色光蛋白 (eGFP) 中,测试了标签的适用性.
  • 经过验证的标签功能与修改的GroEL陪伴域 (GrAD207).

主要成果:

  • 成功地将内部His标签纳入eGFP和GrAD207.7中.
  • 证明与终端标签相比,内部标签可以最大限度地减少硬体障碍.
  • 显示了在不同蛋白质类别中广泛适用的潜力.

结论:

  • 内部His标签为N/C终端融合提供了一个可行的替代方案.
  • 设计的标签可以调整以有效净化各种蛋白质.
  • 这种方法扩大了聚变蛋白的功能,但不损害术语.