Integral membrane proteins constitute a significant fraction of the proteome of all living cells. Important examples of this protein class include certain signal transduction receptors, cell adhesion molecules and ion channels. The biochemical and structural characterization of this challenging class of protein represents an important frontier in both basic science and drug discovery research. However, due to their unique physical properties and requirement for association with cellular membranes, expression of membrane proteins in heterologous systems is often daunting.
Cellular expression of recombinant membrane protein often results in protein aggregation and misfolding due to the hydrophobic nature of transmembrane segments. When using E. coli as a host, it is advantageous to express membrane proteins in moderation to avoid saturation of the membrane protein biogenesis pathway, which may lead to cell death and/or inclusion body formation. The Lemo21(DE3) protein production strain was designed for tunable T7 expression to achieve optimal assembly of transmembrane proteins or the optimal folding of soluble proteins. In standard T7 expression strains (e.g. BL21(DE3)) transcription of a target gene from a T7 promoter may be too robust and it is not easily controlled. In contrast, Lemo21(DE3) expresses a T7 RNA polymerase inhibitor protein (LysY) so that the level of target gene transcription may be precisely regulated. Lemo21(DE3) allows researchers to sample a wide range of expression levels to find the optimal conditions for each unique target protein. In the case of membrane proteins less expression often results in more functional protein.
- 5 Minute Transformation Protocol (C2528)
- 5 Minute Transformation Protocol (C3013)
- Affinity Purification and On-column Cleavage (E6901)
- Cell Surface Labeling ACP-Surface Starter Kit
- Cellular Labeling (E9120)
- Cellular Labeling (E9230)
- Cleavage of the Fusion Protein Generated Using The pMAL Protein Fusion and Purification System (E8200)
- Cloning a PCR Fragment Into a pMAL Expression Vector (E8200)
- Construction of the Fusion Plasmid (E6901)
- Expression Using SHuffle (C3026)
- Fusion Constructs (E6901)
- Fusion Protein Expression (E6901)
- High Efficiency Transformation Protocol
- High Efficiency Transformation Protocol (C3013)
- Labeling of Proteins in Solution (E9230)
- Labeling of Proteins in vitro for ACP-Surface Starter Kit
- Labeling Proteins in vitro (E9120)
- Preparation of Media and Solutions (E6901)
- Primer Design for Restriction Enzyme Cloning (E6901)
- Protein Expression Using Lemo21(DE3) (C2528)
- Protein expression using the K. lactis Protein Expression Kit - Cloning a PCR fragment into pKLAC2 (E1000).
- Protein Expression using the K. lactis Protein Expression Kit - Growth of strains for detection of secreted protein
- Protein Expression using the K. lactis Protein Expression Kit - Identification of Multi-copy Integrants
- Protein Expression using the K. lactis Protein Expression Kit - Identification of properly integrated cells
- Protein expression using the K. lactis Protein Expression Kit - Linearization of pKLAC2 for integrative transformation of K. lactis.
- Protein Expression using the K. lactis Protein Expression Kit - Simultaneous Expression of Multiple Proteins
- Protein Expression using the K. lactis Protein Expression Kit - Transformation of K. lactis GG799 cells
- Protein Expression with T7 Strains
- Protocol for Expression Using T7 Express lysY/Iq (C3013)
- Purification of a Fusion Protein generated by The pMAL Protein Fusion and Purification System (E8200)
- Quick Start Guide (E8200)
- Separating the Protein of Interest from MBP after Protease Cleavage Using The pMAL Protein Fusion and Purification System (E8200)
- Simplified Expression and Purification Protocol (E6901)
- Transformation Protocol (C2528)
- Use of CLIP-Cell Block with CLIP-Cell Substrates (E9230)
- Western Analysis (E8023)
Avoid Common Obstacles in Protein Expression
Read how to avoid common obstacles in protein expression that prevent interactions with cellular machinery.
- Competent Cells Brochure
- Competent Cell Product Comparison
- Convenient Formats of Competent Cells
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NEB has a long history in recombinant protein expression and has developed a wide array of solutions for proteins that are difficult to express.