Many proteins are extremely difficult to express in heterologous expression systems. A vast number of factors may contribute to this problem. A common problem is that it can often be challenging for a foreign host to correctly fold a protein it does not normally produce. For example, in many experimental scenarios expression of a protein originating from a higher eukaryote is being produced in a bacterium where factors such as codon usage, translation rate, and redox potential are significantly different. Additionally, inherent properties of the target protein may represent challenges for the expression host. For example, a protein having multiple membrane spanning domains might not properly insert into membrane bilayers of the heterologous host or a protein might not be expressed in a soluble form. Finally, many proteins require post-translational modifications (e.g. glycosylation or phosphorylation) that are absent or significantly different from expression host to expression host.
There is no single solution for the expression of all classes of difficult proteins. Instead, expression problem-specific solutions that aim to better the chances of success can be used. For microbial expression systems, these solutions often come in the form of unique host strains that have been genetically modified to enhance the production of a certain difficult protein class. Other expression solutions seek to address problems by controlling aspects of how a target protein is produced. For example, some expression hosts allow for precise control of target gene expression. In addition, certain protein tags can help a protein to more efficiently insert into a host membrane or improve the solubility of a target protein.
Expression of Difficult Proteins includes these areas of focus:
FAQs for Expression of Difficult Proteins
- What applications are SHuffle® strains useful for?
- Which SHuffle® strain should I use?
- How do SHuffle® strains aid in cytoplasmic disulfide bond formation?
- What is LysY?
- K. lactis Protein Expression FAQs
- What are the strain properties of Lemo21(DE3) competent E. coli?
- What systems does NEB offer for protein expression and purification?
Protocols for Expression of Difficult Proteins
Application Notes Expression of Difficult Proteins
- E. coli Lemo21(DE3)
- Protein Expression with T7 Express Strains
- Transformation of SHuffle® Competent Cell Strains
- Use of the PURExpress® in vitro Protein Synthesis Kit, Disulfide Bond Enhancer and SHuffle® Competent E. coli for heterologous in vitro and in vivo cellulase expression.
- Using the PURExpress® In Vitro Protein Synthesis Kit for Heterologous In Vitro Expression and Functional Screening of FMN-dependent Oxidoreductase Variants
Bypassing Common Obstacles in Protein Expression
The Next Generation of Cell-free Protein Synthesis
Competent Cells Brochure
The Competent Cells brochure provides information on the different competent cell strains for cloning and protein expression available from NEB.
Protein Expression & Purification Brochure
The Protein Expression and Purification brochure provides information on the advanced tools for protein expression and purification offered by NEB.
- Competent Cell Product Comparison
- Protein Expression and Purification Selection Chart
- Convenient Formats of Competent Cells
Other Tools & Resources
T7-Controlled Expression of Protein in E. coli Hosts
Improvement of Protein Solubility with Lemo21(DE3)
The Lemo System™ for Membrane Protein Expression
Disulfide Bond Formation
PfCHT1 Chitinase Expression in SHuffle® T7 Express
vtPA Expression in SHuffle®
Improved Purity of His-Tagged Proteins with NiCo21(DE3)
NiCo21(DE3) Two-Step Purification
Optimization of YidC-GFP Expression with Lemo21(DE3)
Lemo21(BE3) vs. BL21(DE3)
Protein Expression Using the PURExpress® In Vitro Protein Synthesis Kit
Schematic Illustration of Purification Using pMAL™ System
Protein Expression Using pMAL™
NEB has a long history in recombinant protein expression and has developed a wide array of solutions for proteins that are difficult to express.