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Tagging and Fusion Proteins

Tagging and Fusion Proteins

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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Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions

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Tools to cope with difficult-to-express proteins.

Paolo Saccardo^1,2, José Luís Corchero^1,2,3, Neus Ferrer-Miralles^4,5,6

¹Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, 08193, Barcelona, Spain.

Applied Microbiology and Biotechnology

|April 16, 2016

Summary

Recombinant protein production faces challenges with difficult-to-express genes in systems like Escherichia coli. This review proposes an integrative approach to optimize protein expression and purification processes for better yields and activity.

Keywords:

Biopharmaceuticals Difficult-to-express protein Escherichia coli Expression system Protein aggregation Protein solubility Recombinant protein production

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Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Published on: June 20, 2019

Area of Science:

Biotechnology
Molecular Biology
Protein Engineering

Background:

High-throughput genomics and recombinant DNA technology have advanced protein production.
Recombinant biopharmaceuticals represent a multi-trillion dollar global market, indicating sustained demand.
Escherichia coli is a cost-effective expression system but often struggles with expressing complex or problematic genes.

Purpose of the Study:

To address challenges in producing difficult-to-express recombinant proteins.
To propose an integrative strategy for optimizing protein production and purification.
To rationalize efforts in recombinant protein expression by considering pivotal factors.

Main Methods:

Review of existing literature on recombinant protein expression systems and challenges.
Analysis of factors influencing protein expression, solubility, and activity.
Discussion of an integrative approach to process design, moving beyond trial-and-error.

Main Results:

Many genes are not efficiently expressed in common systems like E. coli, leading to protein aggregation, degradation, or loss of function.
Alternative homologous systems can improve expression but increase complexity and cost.
A growing toolbox of techniques exists, but their application is often uncoordinated.

Conclusions:

An integrative, knowledge-based approach is needed to rationally design recombinant protein production processes.
Optimizing expression requires considering multiple factors across the entire production pipeline.
This strategy aims to improve the efficiency and success rate of producing challenging proteins.