Protein Co-expression in E. coli

In organisms, many proteins are active only as heteropolymer complexes. Therefore, detailed biophysical, biochemical and structural studies depend on the preparation of protein complexes in milligrams. Among all the expression systems, bacterial overexpression system is still the most convenient one. The preparation of complexes usually depends on the ability to reconstruct such protein complexes from separately prepared recombinant proteins, which usually involves renaturation. At Creative BioMart, we developed a recombinant protein co-expression system. The system uses two T7 promoter-based vectors, which can stably co express binary protein complexes in bacteria.

Technology Description

Our co-expression system involves 2 T7 promoter-based vectors, which can stably co-express binary protein complexes in bacteria. The two vectors have different replication starting points to allow the cells to support both expression vectors at the same time, and to allow the selection of different antibiotic resistance of the cells containing the two plasmids. Another advantage of using two independent plasmids is that they can mix and match different protein structures, greatly simplifying the optimization of different heterologous protein combinations.

Expression vector 1

It is a plasmid with a very high copy number. It contains an ampicillin resistance gene and is the starting point of pUC replication. There is a 6xHis tag in the MCS, which can be added to one or more components of the complex. The vector can also be used for initial expression, solubility and purification of the target protein.

Expression vector 2

Vector 2 is a low copy number co-expression vector designed with vector 1. It contains M13 replication starting point, kanamycin resistance gene, T7 promoter, ribosome binding site, 6xHis fusion tag, MCS and T7 termination sequence from vector1.

Protein Co-expression in E. coli

What Can We Do For You?

We offer a variety of flexible service packages for our clients.

Service Name	Service Description
Preparation of target DNA	Codon optimization Plasmid purification Gel purification
Subclone construction	Cloning of protein constructs into vector 1 Test for protein overexpression on a small scale Cloning of protein constructs from vector 1 into vector 2 Test protein expression from vector 2 Prepare competent E.coli cells harboring vector 2 containing the cloned protein sequence of interest
Protein expression	Transform the cloned vector 1 into competent cells that contain vector 2 Co-expression of proteins The cellular and subcellular components, expression time, temperature conditions, protein solubility and activity test Qualitative and quantitative analysis of protein products
Protein purification	Amplification culture Preparation of crude extract Affinity purification Cut off fusion tag and remove protease (if necessary)

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