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Recombinant Protein Expression in E coli Services

2016-11-30
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E. coli Expression System Services

 

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.

 

Service:

  1. Generation of Recombinant Plasmid

  2. Expression Test in Host

  3. Soluble Expression Test

  4. Small Scale Expression and Purification (2L Cell Culture)

  5. Scale Up Expression and Purification

StepsItemsTimelineComments
1Generation of Recombinant Plasmid2 weeksMultiple expression vector, different affinity tag: His, Trx , GST, SUMO…
2Expression of Recombinant Protein1 weekMultiple expression host
3Soluble Expression Optimization1-2 weeksTest different induction concentration, time point, temperatures or reconstruct plasmids
4Small Scale Expression and Purification1-2 weeksPurification of soluble expression or from inclusion body
5Scale Up Expression and PurificationTBDCulture in flask or fermenter

Remarks:

  1. The service is highly flexible to meet your request. One or more above steps could be separately provided.

  2. 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.

Why Express in E. coli?

  • 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.

advantage of e coliGeneral Background for Protein Expression in E. 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.

What are the important features of gene expression in E. coli?

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.

Contact Us 

Email : marketing@medicilon.com
Tel : +86 021 58591500

Tips : Above is part of Protein Expression in E coli Services, Recombinant protein expression in E.coli . You can also CONTACT US with any question or enquiry you may have. We will be happy to discuss your needs in detail and design an appropriate plan of action.

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The prokaryotic expression system (E. coli expression system)

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