Expression Of Recombinant Proteins In Eukaryotes

Introduction:

During last two centuries, the expression system of prokaryotes is being used in order to produce large amount of recombinant proteins. The challenges during this production that are being faced by biotechnologists in order to maintain balance between the amount and quality of these proteins. All of these challenges will be discussed in this article.

 

In case of prokaryotic expression system the prokaryotic cell that is preferred is E.coli and the reasons of preferring this cell are as follows:

 

·         Carbon source is required for growth which is inexpensive.

·         Biomass is accumulated rapidly.

·         This process is very simple.

 

However, in this case there is deficiency of post translational machinery and inactive proteins may also be formed as a result of the formation of inclusion bodies. On the whole we can divide these strategies into three categories:

 

Group 1: The factors that are responsible for the development of insoluble fraction, they are altered by tightly controlling cellular milieu, thus forming expression of soluble protein.

 

Group 2:In this the protein that is expressed is again folded from inclusion body.

 

Group 3:In this case the expression that is desirable is obtained in soluble form.

Before describing this detail, there are various environmental factors and genetic factors that are included in expression systems of bacteria, that are responsible for influencing the successful production of recombinant proteins.

Most of the amino acids are encoded by same codon and all codons of amino acids are used for each organism. The codon bias of mRNA is showed by its tRNA [30,31]. In case of E.coli translation rate is lowered during over expression of target gene and therefore more than one rare tRNA is required and also this occurs due to change use of codon [32-34].

If we are using expression systems of E.coli that are of high level, than if rare codons are present than they are unable to reduce the synthesis of target protein. However, this situation is reversed when large amount of rare codons are present, they are over expressed in this situation and they cannot reduce the synthesis of target protein [1].

Expression of protein:

Mature technology is the expression of proteins by using different vectors like bacteria. In order to produce expression plasmids we can use the techniques of PCR and cDNA. We have to transform plasmids into the expression hosts and then pick the single colonies and then perform fermentation.

If the host is E.coli and our fermentation media is 2 liter than the resultant weight will be 50 to 80g. The modest expression of protein is obtained between 100 and 300mg. The problem in this case is the separation of recombinant proteins and also retaining its activity. If the proteins are soluble than the resultant yield will be high but if the proteins are in insoluble form than they have to pass through the steps of denaturation and refolding.

If we just want to determine the structure of proteins and then just laboratory equipments and small scaled fermentation is enough to obtain the desirable concentration. There are some scientists who had reviewed the production of proteins on large scale in E.coli.

Same is true for the production of proteins in other eukaryotic expression system but more effort is required in order to construct vectors. Those proteins that form insoluble inclusions, the best host for these proteins is yeast. In order to produce glycoproteins and phosphorylated proteins than baclovirus system is best for this purpose.

More efforts are required if we want to construct expression system for mammalian proteins. If the density of cell is 1-5 x 10⁹ than the final product obtained from this cell would be greater than 10mg. The small amount of proteins can be produced by using viral vectors if they are just required for study purpose.

Many biotechnology companies develop expression systems for the large scale production of recombinant proteins [Wurm and Bernard, 1999]. E.coli is the best expression system if we want to obtain recombinant proteins for investigation in laboratory. E.coli is preferred because it can easily grow and expression plasmids are easily available.