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Related Concept Videos

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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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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.
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Tail-anchoring of Proteins in the ER Membrane01:45

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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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Mitochondrial precursors are translocated to the internal subcompartments via independent mechanisms involving distinct protein machineries called translocases.
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Signal sequences are short amino acid sequences that guide newly synthesized proteins to their proper location within the cell. Classical signal sequences are fifteen to sixty amino acids long and present at the N-terminus of a polypeptide chain. Each signal sequence has a conserved segment of basic residues towards their N terminus, a hydrophobic core, and a C-terminus rich in polar residues. The C-terminus also contains a signal cleavage site and features a -3 -1 sequence motif. The -3-1...
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The ubiquitin-proteasome pathway is a well-known mechanism utilized by eukaryotic cells to remove cytoplasmic proteins that are misfolded, damaged, or no longer needed. In this pathway, the protein that needs to be eliminated undergoes a process called ubiquitination, where a chain of ubiquitin molecules is attached to the 48th lysine residue of the target protein. This ubiquitin modification helps the proteasome distinguish between a target protein and a healthy protein.
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A cleavable self-assembling tag strategy for preparing proteins and peptides with an authentic N-terminus.

Qing Zhao1, Bihong Zhou1,2, Xianxing Gao1

  • 1Department of Chemical Engineering, Tsinghua University, Beijing, China.

Biotechnology Journal
|March 16, 2017
PubMed
Summary

Researchers developed a new method for protein and peptide purification using self-assembling peptides and inteins. This strategy simplifies purification, yielding proteins with an authentic N-terminus efficiently and economically.

Keywords:
Authentic N-terminusC-terminal cleavage inteinPeptide preparationProtein expression and purificationSelf-assembling tag

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Area of Science:

  • Biotechnology
  • Protein Chemistry
  • Molecular Biology

Background:

  • Recombinant protein and peptide production is crucial for biotechnology.
  • Existing purification methods can be complex and costly.
  • Producing proteins with authentic N-termini is often challenging.

Purpose of the Study:

  • To develop a streamlined and economical strategy for protein and peptide purification.
  • To create a method yielding target molecules with an authentic N-terminus.
  • To improve the purification of unstable peptides.

Main Methods:

  • Utilized a fusion protein strategy involving self-assembling peptides and C-terminal cleavage inteins.
  • Expressed fusion proteins as aggregates induced by self-assembling peptides.
  • Employed intein-mediated cleavage for release of target proteins/peptides.

Main Results:

  • Tested combinations of four self-assembling peptides (ELK16, L6 KD, FK, FR) and three inteins (Sce VMA, Mtu ΔI-CM, Ssp DnaB).
  • Identified Mtu ΔI-CM intein with L6 KD, FR, and FK peptides as an effective combination.
  • Achieved yields comparable to established strategies (e.g., Trx-strategy) with enhanced simplicity and cost-effectiveness.

Conclusions:

  • The novel strategy offers a simple and efficient method for purifying proteins and peptides.
  • This approach is particularly effective for producing unstable peptides (30-100 amino acids) with authentic N-termini.
  • The method simplifies recombinant protein expression and purification processes.