Basically for longer period of time. The Genome of

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 follows

1.
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.

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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.
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 easy

Advantages
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