Basically there are four most common expression systems used for recombinant protein production in eukaryotic cells. Each system has its own features, advantages and limitations which are discussed as follows1. Expression of recombinant proteins in Yeast cells: In recent years’ production of recombinant protein is very rapidly growing in the field of molecular biology and medicine and presently plays very crucial role in curing many diseases. Among many expression systems yeast expression system form recombinant protein production shows very strong growth on simple media, it has capability of quickly acquiring genetic changes and especial it includes Post Translational Modifications. Saccharomyces cerevisiae is the most commonly used host cells for the recombinant protein production. Though, there are other yeast species like Pichia pastoris, Yarrowia lipolytica and Hansenula polymorpha have increasing attraction for production of the recombinant protein.The main features of yeast that attracts the scientist for a better host to produce recombinant proteins are, it is a single cell organism with very short doubling time of 1.3 to 2 Hr.
at 30? C. It is easy to be cultured in large quantity with very low cost, simple easily available solid or liquid medium. It can be stored at -80° C for longer period of time. The Genome of yeast is fully sequence which help us for genetic manipulation for better results of expressing recombinant proteins.
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It is easily transformed with the help of Different plasmid vectors. It has Post Translational Modification system which is very for the eukaryotic recombinant proteins.Advantage of yeast expression system are; it preserves many cellular processes of different eukaryotic species; it has Protein folding mechanism for produced recombinant proteins; also Post Translational Modifications system for maintaining the activity of the produced protein; Yeast cells are very easy and low cost affair for growth with simple media and needs small space, it can secrete uncommon of its own proteins. Recombinant protein can be easily purified. Significant variety of vectors and genetic information is available for better results, along with promoters and regulatory systems. Advantages of production in Pichia pastoris are, it has highly efficient promoter with firm regulation (alcohol oxidase AOX, induced by MeOH).
it has very high cell density and can produce no EtOH. For therapeutic recombinant protein there is no risks of contaminations with human pathogens.Disadvantages of yeast expression system are; it takes more time for growth as compare with bacteria (doubling time for bacteria is 20- 30 min in contrast for yeast it is 1.
5 – 2 hours at 30° C. In yeast cells Hyper glycosylation of produced proteins can be detected. Post Translational Modification like glycosylation results into different changes as compared to actual desired proteins. For some recombinant proteins production levels might be low.
Sometimes it can secret not good proteins. Sometime proteins may have retained in cell wall of host cell. Yeast system give rise to high level production of EtOH which is toxic for the cells.Limitations of yeast expression system are, it shows loss of plasmid vectors during process, specifically when recombinant proteins are harmful in nature to the host cells. Yeast has the capability to attach glycan’s group at both specific asparagine residues (N-linked) and serine/threonine residues (O-linked) in the recombinant protein produced which is totally different in case of post translational modifications occurred in mammalian cells. Expression of the desired recombinant protein in Pichia pastoris involves methanol. During large-scale production, the high quantity of methanol becomes a fire hazard, which lead to get more safety conditions.2.
Expression of recombinant proteins in Insect cell:Among the varied choice of expression system present for the production of recombinant proteins, insect cell expression system is one of the best for the production of complex proteins demanding general post translational modification. The main stream proteins produced by eukaryotes are glycosylated. Though, one should know and has an interpretation that a proteins post transcriptional modification arrangement is definite for each species, tissue and cell. transcriptional modification is not always essential for the activity of the desired protein, but there are different report shows that it is essential.
Sometime excessive, Hyper glycosylation can result into undesirable activity of the protein to be produced. Insect cells have ability to perform more complex post translational modifications as compare with the yeasts, along with not only glycosylation, but also acylation, phosphorylation, carboxy methylations, correct proteolytic processes, signal peptide processing. Insect cells moreover practice disulphide bonds more correctly and are able to give high levels of expression. Additionally, they have the finest mechanism available for mammal protein folding except from mammals themselves.
The Insect cells are transformed with the help of baculovirus vectors, they are naturally pathogenic in nature. This type of vector can easily transfect the host cells with high rate. Because of polyhedrin a strong promoter insect vector has advantage.
Feature of insect cells for recombinant protein production are, it shows high level expression through the last phase of lytic cycle prior cell lysis. This expression system is appropriate for production of intra cellular as well as extra cellular proteins. This system can efficiently produce disulphide bonds in desired proteins. It provides all major post transcription modification in mammalian cells.Advantage of this system are, mostly all the protein produced by this system is active in nature and soluble. This system can give very high level expression of desired protein up to 50% which very impressive and this is because of strong promoters. Almost post translational modifications are similar to mammalian cells.
The viruses’ vectors used are very specific and they are not harmful to mammals. It shows very good level of expression for intracellular proteins and comparatively fast growth with efficient protein folding. It is moderately scalable; it is endotoxin free. Disadvantages of this system are, it is very expensive affair as culture media costly.
It requires large quantity of virus for scale up. Sometime viral infection may cause cell lysis, disruption and loss of desired protein. sometime insufficient post translational modification might occur which lead to different protein secretion and folding of protein.
Limitations of insect cell expression system are, many time the glycosylation in this system is different than the desired protein hence undesired recombinant protein will produce, in this system large fraction replication is poorly processed and hence aggregation of proteins occur unfolded protein cluster form. Sometime discontinuous expression or desired protein occurs which lead to low level expression of protein. It is inefficient for industrial level scale up. 3. Expression of recombinant proteins in Mammalian cell:In recent years, the biopharmaceutical industry has expressively turned its recombinant protein production to mammalian cell expression systems.
The capability of post translational modification mechanisms in these systems are key features of this system. And in the recent day’s importance of monoclonal antibodies in medicine field has attracted this system very essentially. Amongst the mammalian expression system, CHO cell line is the most regularly used cell line. CHO cell line lonely produces up to 70% of recombinant proteins, among them most are monoclonal antibodies. Other commonly used mammalian cells are 1(COS) Simian fibroblasts, (NEK293) Human embryonic kidney cells.
Baby Hamster Kidney (BHK) cell line is used for the production of vaccine. New technology like omics has given us new opportunities and chances of improvement in these expression system.Features of mammalian expression system are, it gives specific recombinant protein production with perfect post translational modification and they are present in their bioactive forms. This system gives ephemeral and steady expression of recombinant protein.Large scale recombinant protein production is possible. Modular multidomain arrangement is present. This system forms disulphide bridges.
Glycosylation of desired protein is done in a such fashion so that protein produces in natural state with their native structures, chemical characteristics and activity. Currently this system involves in production of biopharmaceutical like monoclonal antibodies, cytokines, growth factors and many more. This system shows fast and easy transient expression. This system can give very stable transfection which lead to more productivity.Advantages of mammalian expression system are, All the desired protein produces have most accurate post translational modifications. All proteins are folded in their native forms. This system can produce both secretive protein and membrane protein.
It gives us reasonably fast expression of desired protein as compare to other expression systems. This system gives highest number of functionally active recombinant protein compart other expression system. Recombinant protein produced by this system shows high compatibity with the humans and also they show very low immunogenicity to humans. Protein produce by this expression system is highly safe to use and gets easy permission to use it as a drugs against human diseases. The main disadvantages of this system is, it is very expensive affair for experimentation, final yields of intracellular proteins are low, final product is very difficult to scale up as there are many by product.
Transfection of our gene of interest into used mammalian cells is very hard. The entire procedure is very time consuming.limitation of mammalian expression system, it is well known that mammalian expression system gives functionally actively desired proteins because of its post transcriptional modification mechanisms. Despite of these advantages, this system has limitations like It is very expensive experimental process, the working technology is complicated, needs very skilled staff for practice, It has probable chances of contamination with foreign viruses, which is not safe to practice also affect desired protein 4.
Expression of recombinant proteins in Plants:Plants deliver varied choice of significant biological molecules that are desirable in research, medicine and industry. Presently, plants as expression system has increasing attention for recombinant protein production. The recombinant protein production in plant is safe there safe, cost-effective it can be easily scaled up compare to microbial and other expression system and there are no ethical issues.
Though plant systems are accepted for producing recombinant proteins but there are many problems with this system which need to be modified and improved like promoters, protein stability, activity and purification. The main reason for which attract this system is it can be easily transformed, very low cost sources and not hazardous. Specific recombinant proteins with desired characteristics are generally expressed in transgenic plants. Commonly transgenic plants are manufactured by two methods, the first one is Agrobacterium mediated transformation and the second one is standard transformation techniques. There are many recombinant proteins which are produced by plant expression system such as Phytase, Hirudin, Papain, Monelin, Enkephalin and thaumatin.
Features of plant expression system are it is very safer production method, cheaper than the other systems, sometime no need of purification as plant parts are edible so we can directly consume our desire proteins. There is no ethical problem with this system. More profitable than the other systems, cause no need of special storage container or purification machineries. The scale up for this system is very easyAdvantages of plant expression systems for production of recombinant proteins are, the plant are more plants are better in terms of safety, handling, cultivation and distribution as compared with animals, microbe and animal cell line. The other factors which is considered that plant are more superior compare to other system are cost, consume less time and protein complexity. With this system there is very less chance of contaminations because no plant diseases have been found harmful to the humans. Recombinant protein can be produced in plats part which are edible so that it will remove the purification process and cost will be reduced.
Plants can also perform most of post translational modifications which are needed for protein folding and stability.Limitation of plants expression systems are, for the production of therapeutic proteins in plant is still under developing stages. Limitations rises in this system because low level expression of desired protein, clustering of desired protein and improper post translational modifications of the recombinant protein. Plant system have deficiency of terminal galactose and sialic acid residues which are commonly found in animals and does have ?-(1,3) fucose and ?-(1,2) xylose which are lacking in animals, therefore this will lead to the affecting the properties of desired recombinant protein. Other limiting factors arises from the of proteins expressed in plants.
The post translational modifications show changes in glycosylation, folding and proteolytic processing which might affect the activity, function and stability of desired recombinant proteins