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Mempro™ Protein Production in Bacterial Cells System

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Creative Biolabs can offer advanced Mempro™ membrane protein production services using bacterial cells system. We can obtain the highest production of your target membrane protein using this high-quality system.

Escherichia coli (E. coli) is by far the most widely applied bacterial host for the production of membrane proteins. It is reported that a derivative strain of E. coli, termed Lemo21 (DE3), is engineered in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7 Lys). In Lemo21 (DE3) strain, a large amount of recombinant membrane protein can be acquired easily. The development of this strain is sped up tremendously by using GFP-fusions to monitor membrane protein production. Strikingly, the Lemo21 (DE3) strain can be used to produce membrane protein GFP-fusions from which properly folded membrane protein will be recovered for further functional and structural studies (Figure 1).

Mempro™ Protein Production in Bacterial Cells System Figure 1. Genal procedures of membrane protein production yields in E. coli cells. (Biochim. Biophys. Acta., 2014)


Creative Biolabs employs Lemo21 (DE3) strain for Mempro™ protein production in E. coli system. In this optimized system, expression of the gene encoding the T7 RNA polymerase is governed by the isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible lacUV5 promoter, and expression of the gene encoding the T7 lysozyme (T7 Lys)-the inhibitor of the T7 RNA polymerase, is governed by the rhamnose promoter from the pLemo plasmid. The gene encoding the target membrane protein is located on a pET vector, which is governed by the T7lac promoter. As a result, membrane proteins are expressed as C-terminal GFP fusions. The GFP moiety only folds properly and becomes fluorescent when the membrane protein GFP fusion is inserted in the cytoplasmic membrane. In the next step, Lemo21 (DE3) cells are cultured in the presence of different concentrations of rhamnose. The expression of genes encoding membrane protein-GFP fusions is induced with IPTG. Whole-cell fluorescence is used to monitor the production of membrane protein-GFP fusions in the cytoplasmic membrane, in order to assess the integrity of the produced membrane protein GFP-fusions. The ratio of the cytoplasmic membrane inserted to non-inserted membrane protein is monitored using an SDS-PAGE/immuno-blotting based assay. The fluorescence assay facilitates the determination of a detergent to optimally solubilize the membrane protein from the membrane, and to monitor the stability of a membrane protein in a detergent. Finally, membrane proteins can be recovered from the GFP-fusion using a site-specific protease and subsequently be used for functional and structural studies.

Besides E. coli, Ceative Biolabs can select other bacterias as membrane protein production hosts, such as Lactococcus lactis (L. lactis), Bacillus subtilis (B. subtilis), and Rodhobacter sphaeroides (R. sphaeroides). B. subtilis or L. lactis bacteria, express two copies of the integral membrane chaperone YidC, can perform better at insertion and assembly of heterologous membrane proteins than E. coli. While R. sphaeroides is a Phototrophic bacteria, the main feature of this bacteria as the membrane protein production host is hight yield of membrane proteins.

Creative Biolabs can provide other various Mempro™ membrane protein production services. Please feel free to contact us for a detailed quote.

References:
N. Ferrer-Miralles, et al. (2013). Bacterial cell factories for recombinant protein production; expanding the catalogue. Microb Cell Fact., 12: 113.
P. D. Laible, et al. (2004). Towards higher-throughput membrane protein production for structural genomics initiatives. J. Struct. Funct. Genomics, 5(1-2): 167-72.
S. Schlegel, et al. (2014). Bacterial-based membrane protein production. Biochim. Biophys. Acta., 1843(8): 1739-1749.




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