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Practical Tips | Guide to Choosing a Protein Expression System

Release time:

2025-01-03

What is a Protein Expression System?

Protein expression refers to a molecular biology technique that uses hosts like bacteria, yeast, animal cells, or plant cells to express foreign gene proteins.

A protein expression system is a setup composed of a host, foreign gene, vector, and auxiliary components, which enables the expression of the foreign gene within the host.

Classification of Protein Expression Systems

Expression System	Host Organism
Cell-free Expression System	None
Prokaryotic Expression System	Escherichia coli
Prokaryotic Expression System	Bacillus subtilis
Eukaryotic Expression System	Yeast cells
	Mammalian cells
	Baculovirus-Insect cells
Transgenic Animal/Plant Systems	Transgenic animal/plant bioreactor

Mainstream Protein Expression Systems on the Market

Currently, the mainstream protein expression systems on the market include the prokaryotic Escherichia coli expression system as a representative, as well as eukaryotic expression systems such as yeast cell expression systems, mammalian cell expression systems, and baculovirus–insect cell expression systems.

Protein Expression System Selection Guide

When choosing a protein expression system, the biological characteristics of the target protein and the planned downstream applications determine the most suitable expression system. Therefore, it is important to gather information about the natural localization of the target protein (intracellular, secreted protein, or membrane protein), size/molecular weight, whether it is a single-domain or multi-domain protein, the number of disulfide bonds, post-translational modifications (such as glycosylation), and any auxiliary factors needed for correct folding and structural integrity. This information can be gathered through literature, databases, or bioinformatics tools.

Once the biological characteristics of the target protein are collected, four key questions can help determine the optimal expression system for the target protein:

Do you want to produce prokaryotic or eukaryotic proteins?

Does the downstream application require post-translational modifications? (Does it involve disulfide bonds, protein/protein complex molecular weight, or the complexity of membrane protein integration?

What type of glycosylation is required?

Is higher yield or multiple rounds of expression needed?

Typically, Escherichia coli expression systems are preferred for the production of prokaryotic proteins, although other bacterial protein expression systems are also available. For eukaryotic protein production, several factors play a role in the decision-making process. For simple eukaryotic proteins that do not require post-translational modifications and have a limited number of disulfide bonds, Escherichia coli can also be used as an expression host. However, in many cases, eukaryotic expression systems such as yeast, insect cells, or mammalian cells may be more suitable. One of the main differences between these eukaryotic hosts is the type of glycosylation they provide.

Nanjing Pronetbio Business Introduction

Nanjing Pronetbio offers Escherichia coli expression systems, yeast cell expression systems, and mammalian cell expression systems. Each of these protein expression systems has its own distinct advantages.

System Comparison

Expression System	Prokaryotic Expression System	Yeast Cell Expression System	Mammalian Cell Expression System
Host Organism	Escherichia coli	Yeast cells	Mammalian cells
Advantages	1.Clear genetic background, multiple recombinant vectors and tags available. 2. High expression levels, efficient protein synthesis. 3. Fast cycle time.	1. Some post-translational modification capabilities. 2. Stable production process, suitable for large-scale production. 3. No endotoxins, high safety. 4. Relatively high expression levels.	Complete post-translational modifications, yielding recombinant proteins with structures and modifications nearly identical to native proteins.
Disadvantages	1.No post-translational modifications. 2.Prone to inclusion body formation. 3.Difficult to express high molecular weight proteins. 4.Endotoxin presence.	Some probability of misfolding and incorrect modifications.	Complete post-translational modifications, yielding recombinant proteins with structures and modifications nearly identical to native proteins.
Applications	Suitable for simple proteins with low post-translational modification requirements.	Suitable for proteins requiring some degree of post-translational modifications.	Suitable for proteins with stringent post-translational modification requirements, such as therapeutic proteins.

Q&A

Why is codon optimization necessary?

In most genomes, there is a preference for the use of synonymous codons. When the preferred codons of the target protein are used for translation in the host organism, it will inevitably impact the efficiency of expression. Codon optimization refers to using the most commonly used codon set in the host cell to express the target protein, without altering the amino acid sequence, in order to enhance the expression levels of the target protein.

Why should the signal peptide be removed during heterologous protein expression?

First, it is uncertain whether the original signal peptide will function properly in the host organism. Secondly, the vectors used for heterologous protein expression often carry a signal peptide that is suitable for protein secretion in the host organism, while the original signal peptide may negatively affect the solubility of the protein in the heterologous system.

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