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Recombinant Protein Expression System

BY: Nidhi Uppangala | Category: Biotechnology-products | Submitted: 2010-08-28 08:23:01
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Article Summary: "Recombinant proteins are the ones which are produced by genetically modified organisms, after inserting the appropriate gene into their genome.Recombinant proteins are expressed or produced in large quantities using expression systems such as E.coli, yeast, filamentous fungi, mammalian cells, insect cells and also plant cells ar.."


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Introduction:

Recombinant proteins are the ones which are produced by genetically modified organisms, after inserting the appropriate gene into their genome. The recombinant DNA technology was discovered by Cohen and Boyer in 1973 by introducing a pike of foreign DNA into the host cell E.coli. Recombinant DNA technology basically needs three tools, namely enzymes, vehicle DNA and passenger DNA.

Recombinant proteins are the basic foundation for research in biomolecular field and also existence of biotechnology industry. By using recombinant DNA technology required proteins are produced in large quantities and these proteins are used in structural study, enzymatic study, Antigen production and also in producing other pharmaceutical compounds.

Recombinant DNA Technology:

This technology has got following general steps

1. Identification and isolation of gene of interest.
2. Construction of recombinant DNA.
3. Many copies of recombinant DNA are produced
4. These recombinant DNA are then inserted into host cell
5. Transformed host cells are then screened

Recombinant Protein Expression System:

Recombinant proteins are expressesd or produced in large quantities using expression systems such as E.coli, yeast, filamentous fungi, mammalian cells, insect cells and also plant cells are used.

Expression System- E.coli:

Advantages:

1. E.coli proliferates very quickly
2. Insertion of desired foreign DNA can easily be inserted into E.coli cells
3. Culturing recombinant E.coli is very easy and economical
4. Recombinant proteins are produced in high quantities in E.coli cells.

Disadvantages:

1. Post translational modification of these proteins are missing in E.coli system, for example glycosylation of proteins.
2. Recombinant proteins produced using expression system E.coli can be contaminated by endotoxins.
3. Due to the presence of inclusion bodies purification of these recombinant proteins becomes difficult.
4. Functional proteins are produced in a very small quantity.


Expression System - Insect Cell:

Advantages:

1. Very easy to infect these insect cells.
2. Integration of DNA of interest is very stable.
3. Purification of recombinant protein produced by insect cells are very easy as these cells lyses by themselves after every 96 hours due to the presence of virus.
4. Reliable protein folding are also seen.
5. Recombinant proteins are produced in large quantity.


Disadvantages:

1. Post translational modifications are done but they are not as complicated or precise as seen in human cells.
2. Viral proteases present in the recombinant insect cells degrade the protein of interest
3. Optimization is required before doing viral transfection.


Expression System - Mammalian cells

Advantages:

1. Post translational modifications of this recombinant protein are performed. For example glycosylation and phosphorylation are performed in these mammalian cells after the production of recombinant proteins.
2. Highest functionality of recombinant proteins is seen, because of the presence of post translational modifications.
3. Recombinant proteins produced using mammalian cells as expression system show very high compatibility to humans.
4. Recombinant proteins produced using mammalian cells as expression system show very low immunogenicity to humans.
5. Mammalian cells if used are other than the human cells, these cells are not susceptible to human pathogens.
6. Recombinant proteins produced using mammalian cells as expression system is highly safe to use.
7. Recombinant proteins produced using mammalian cells as expression system gets easy permission as a drug to use against human diseases.


Disadvantages:

1. Transfection of gene of interest into these mammalian cells are so far difficult to achieve.
2. When compared with other expression systems production rate of recombinant proteins are very low.
3. Selection of single clone is very time consuming process when compared with other expression systems.
4. Mammalian cell culture technique is expensive.

Advantages of Recombinant Proteins:

1. High percentage of purity.
2. Recombinant proteins show higher specific activity.
3. Continuous supply
4. High level of consistency is seen in batch to batch production of recombinant protein.


Recombinant Protein Products:

1. Hormones: Recombinant DNA technology is used to produce some of the pharmaceutically important hormones such as Insulin, Human Growth Hormone and Erythropoietin.

2. Blood Clotting Factors: Recombinant DNA technology is used to produce some of the pharmaceutically important blood clotting factors like Coagulation factor VIII and Coagulation factor IX.

3. Immunization agent or Vaccines: Recombinant DNA technology is used to produce life saving vaccines such as Hepatitis B vaccine.

4. Recombinant DNA technology is also used to produce large quantities of enzymes which are used in research field.

5. Recombinant DNA technology is also used to produce Interferons.

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Comments on this article: (1 comments so far)

Comment By Comment
farid sajid
2013-06-12 11:48:57 646
Thank you for the info. My question is of a practical nature, Genenetech for example, produces Insulin - my question is: how much recomb DNA. is necessary to produce how much insulin? Sorry if this is a bit vague - I envision a flask of recomb DNA...from which they extract the target molecule - probably very simplistic, but I hope you get the picture.rn So - let's say one 'average dose' ow much of the beginning material would be required to produce that amount? Thank you for your attention and time.

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