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Protein Complexes with Interchangeable Parts

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Groups of proteins may form a complex where each protein in this complex has a different role in the overall execution of the complex’s function. Often some of the proteins in the complex can be replaced by a closely related variant to give a complex that contains many of the same components yet is functionally distinct.
The SCF ubiquitin ligase is a protein complex of five individual proteins. This complex attaches ubiquitin to other target proteins to mark them for degradation. In order...

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Cell-Type Specific Protein Purification and Identification from Complex Tissues Using a Mutant Methionine tRNA Synthetase Mouse Line

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Targeted In Situ Protein Diversification and Intra-organelle Validation in Mammalian Cells.

Mutlu Erdogan¹, Arne Fabritius¹, Jérome Basquin²

¹Tools for Bio-Imaging, Max-Planck-Institut für Neurobiologie, Am Klopferspitz 18, Martinsried 82152, Germany.

Cell Chemical Biology

|March 7, 2020

Summary

Researchers developed a new method using CRISPR/Cas9 to engineer protein variants within mammalian cells for specific functions. This approach enables the selection of improved proteins, like a pH-resistant fluorescent protein, for cellular applications.

Keywords:

CRISPR/cas directed evolution fluorescent proteins mammalian cell organelle targeting protein engineering

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Inducible LAP-tagged Stable Cell Lines for Investigating Protein Function, Spatiotemporal Localization and Protein Interaction Networks

Inducible LAP-tagged Stable Cell Lines for Investigating Protein Function, Spatiotemporal Localization and Protein Interaction Networks

Published on: December 24, 2016

Area of Science:

Biotechnology
Molecular Biology
Cell Biology

Background:

Phenotypic selection is crucial for functional protein engineering in cellular environments.
Existing methods may lack the precision for generating diverse protein libraries within specific cellular compartments.

Purpose of the Study:

To present a versatile in situ protein engineering method for mammalian cells.
To enable the generation and selection of diversified protein libraries in specific subcellular locations.

Main Methods:

Adaptation of CRISPR/Cas9 gene editing technology for in situ protein diversification.
Generation of mammalian cell panels expressing single-cell variants of heterologous protein libraries.
Stable expression of engineered proteins in the cytosol or subcellular compartments.

Main Results:

Demonstrated successful in situ engineering of protein variants within mammalian cells.
Developed a method for intra-lysosome specific selection of engineered proteins.
Identified an extremely pH-resistant, long Stokes shift red fluorescent protein variant.

Conclusions:

The presented approach allows for tailored protein properties for specific cellular compartments and organelles.
This method is valuable for optimizing protein-based tools and biosensors for distinct cellular functions.
In situ protein engineering enhances the development of novel biotechnological applications.