E. coli expression system is the earliest developed and most widely used gene expression system. In recent decades, E.coli expression system has been popularly used and developed for producing a wide variety of different types of proteins. Compared with other expression systems, E. coli is a well-established host with the advantages of short culturing time, easy genetic manipulation and low cost media.
Medicilon researchers established a well-developed E. coli expression and purification services platform, which offers the expression and purification services of a variety of recombinant proteins, with a fast turn-around time and competitive pricing. For more protein expression system,please click on protein expression.
Generation of Recombinant Plasmid
Expression Test in Host
Soluble Expression Test
Small Scale Expression and Purification (2L Cell Culture)
Scale Up Expression and Purification
| Steps | Items | Timeline | Comments |
| 1 | Generation of Recombinant Plasmid | 2 weeks | Multiple expression vector, different affinity tag: His, Trx , GST, SUMO… |
| 2 | Expression of Recombinant Protein | 1 week | Multiple expression host |
| 3 | Soluble Expression Optimization | 1-2 weeks | Test different induction concentration, time point, temperatures or reconstruct plasmids |
| 4 | Small Scale Expression and Purification | 1-2 weeks | Purification of soluble expression or from inclusion body |
| 5 | Scale Up Expression and Purification | TBD | Culture in flask or fermenter |
Remarks:
The service is highly flexible to meet your request. One or more above steps could be separately provided.
Affinity purification, ion exchange purification, hydrophobic beads purification and size exclusion column purification or their combination will be used for protein purification.
Expression of a recombinant protein can be approached in general by constructing a plasmid that encodes the desired protein, introducing the plasmid into the required host cell, growing the host cells and inducing protein expression, and then lying the cells, purifying the protein, and performing SDS-PAGE analysis to verify the presence of the protein.
Well established system of gene expression
Easy to manipulate
Large variety of vectors, strains, methods
Low-tech, safe and inexpensive to grow
suitable for variety of labelings (isotopes for NMR, non-natural amino acids, radioactive)
E.coli tends to be the first choice when expressing heteorlogous proteins or their fragments, be it for antigens, ligand hunting,or structural studies. E.coli offers several advantages and is typically the only bacterial expression system that people try. More complex systems like yeast, insect cells or mammalian cells are usually tried only after E.coli has failed to yield a protein of desired quality.
Prokaryotic expression system (E.coli expression system) is a classical expression system developed earlier and widely used in gene expression technology. In recent decades, E.coli expression system has been continuously developed and improved, and has been widely used by scientific research and industrial users to express various recombinant proteins. Compared with other expression systems, it is characterized by high expression level of target gene, short culture period, strong anti-pollution ability and low cost. Our researchers have established a mature E.coli expression service platform to provide the expression and purification of various recombinant proteins and their complexes in escherichia coli.
An operon is a group of linked genes that are transcribed together, often producing a single message which is then translated into several proteins. The operon structure and the “polycistronic” message allows prokaryotes to coordinately regulate genes that share a common function. In E. Coli, three genes that code for enzymes involved in lactose metabolism, betagalactosidase (lacZ), permease (lacY), and transacetylase (lacA), are produced by the lac operon. These enzymes are normally present at very low concentrations in cells; but when lactose is the sole carbon source, they are induced. After the available lactose is metabolized, the level of RNA transcript and protein returns to a low level. The lac operon is under the control of the lactose repressor protein, encoded by a “cis-acting” regulatory gene, (lacI). The lac repressor asserts negative regulation by keeping the lac promoter inactive in the absence of an inducer.
The repressor functions as a tetramer and binded to a portion of the promoter called the lac operator. Allolactose acts as the inducer in nature. When allolactose is present, it binds to the repressor and prevents the protein from binding to the operator, thus opening up the lac promoter to RNA polymerase.
Gene expression in bacteria, as in all cells, involves
a) the production of messenger RNA by copying of the DNA template by RNA polymerase.
b) translation of the message into protein by the protein synthesis machinery.
In bacteria, these two steps are closely linked. Transcription starts when RNA polymerase binds to the promoter region of the gene, and proceeds to copy the DNA sequence into mRNA.
1. RNA polymerase binding to the promoter region of the gene, followed by transcription to produce a messenger RNA. When the polymerase reaches a terminator sequence, transcription stops, and the mRNA is released from the DNA template. This mRNA contains a 5′ flanking sequence, the actual coding sequence that will be translated, and a 3′ flanking sequence. Because translation does not usually begin at the 5′ end of the message, the mRNAs also carry signals that define the beginning and end of each encoded protein.
2. Initiation of protein synthesis:
The signal that indicates the start site of protein synthesis is usually the AUG, or start codon, which specifies methionine. This site is recognized by the initiator tRNA, which carries a formylmethionine residue.
3. Termination of protein synthesis:
The end of the protein coding sequence is indicated by the presence of a stop codon (UAA, UGA, or UAG). This signal is recognized by release factors, which detach the completed protein from the ribosome.
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The prokaryotic expression system (E. coli expression system)
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Medicilon’s Recombinant Protein Expression Services
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