Yeast Expression System For Glycoengineering Biology Essay

Published:

Translation is a process in which the RNA molecules are translated into proteins. These protein molecules need to undergo several post translational modifications like glycosylation, histone acetylation and deacytylation, addition of hydrophobic groups,etc. These modifications help in making the protein molecule fully functional. This fully functional molecule can further be used in the industry for many purposes. With the advent of the recombinant protein technology, the pharmaceutical companies have been able to exploit this new technology to create various drugs. According to a 2009 survey, it has been reported that global biologic drug sales reached $93 billion, and this figure is expected to rise in the years to come.

Development of new therapeutic agents using genetic engineering requires the help of various expression systems. These expression systems can help mimic conditions of the target patient and thus can help in drug designing. Expression systems can be of the prokaryotic type or of the eukaryotic type. Some examples of the prokaryotic expression system are Escherichia coli and the Bacillus sp., while examples of the eukaryotic expression systems include Mammalian cells, Insect cells, Yeast cells, etc.

Lady using a tablet
Lady using a tablet

Professional

Essay Writers

Lady Using Tablet

Get your grade
or your money back

using our Essay Writing Service!

Essay Writing Service

The European Molecular Biology Laboratory has laid down several guidelines which can help one select a suitable expression system. Some of the guidelines are as follows:-

Type of protein that one wishes to express - The expression system will change depending on the source of the protein - whether prokaryotic or eukaryotic.

Presence of insoluble aggregates when expressed in E.coli - Sometimes, a eukaryotic protein is first expressed in E.coli. This may lead to the formation of insoluble aggregates. Hence, it is necessary to either solubilize the protein with solubilizing agents like Glutathione - S - Transferase or by simply changing the expression system.

Need for post -translational modifications - Eukaryotic and Prokaryotic proteins differ in their post translational modifications, and hence suitable expression systems need to be selected.

Codon usage - There are 61 mRNA codons. Some of these codons are highly expressed, while some are lowly expressed. The lowly expressed codons are called rare codons. The expression of rare codons can be increased by methods such as site directed mutagenesis.

With this short introduction, i would like to focus on the use of Yeast as an expression system in glycoengineering.

Glycoengineering:-

Glycoengineering involves glycosylation of different proteins in order for it to be used as recombinant proteins in the industry. As mentioned above, glycosylation is a type of post translational modification. It involves the addition of oligosaccharides. There are two types of glycosylation - the N - type and The O - type. O - type glycosylation is characterized by the attachment of N - acetylgalatosamine to the serine or threonine residues via an O - glycosidic bond. Further modifications can be made as the protein passes through the endoplasmic reticulum and the Golgi apparatus. N - type glycosylation is very different from the O - type. In N - type glycosylation, a N - glycosidic linkage is formed between the asparagines residue on the nascent polypeptide and the oligosaccharide that has been transferred from the dolichol chain to the N - glycosylation site. The N - glycosylation site has an Asn/X/Ser/Thr, where X stands for any amino acid except Proline.

Glycosylation has many effects on the production of therapeutic proteins. Some of it's effects are listed down as follows:-

Helps in efficient protein folding - FSH and LH have a glycoprotein component that helps in protein folding.

Helps in Protein targeting - Removal of N - glycosylation sites results in a very poor product that cannot be secreted.

Stabilization of protein - Removal of sugar residues triggers aggregation and precipitation.

Regulation of the half life of proteins in the serum - This role is played by the sialic acid that is present in glycosylated proteins.

Helps in ligand recognition and binding - Specific sugar residues on the protein can bind specifically to their receptors on target cells, thus facilitating their uptake.

For these, reasons glycosylation is a very important method of post translational modification.

Yeast as an Expression System -

Lady using a tablet
Lady using a tablet

Comprehensive

Writing Services

Lady Using Tablet

Plagiarism-free
Always on Time

Marked to Standard

Order Now

Characteristics

E. coli

Yeast

Insect cells

Mammalian cells

Cell growth

rapid (30 min)

rapid (90 min)

slow (18-24 h)

slow (24 h)

Complexity of growth medium

minimum

minimum

complex

complex

Cost of growth medium

low

low

high

high

Expression level

high

low - high

low - high

low - moderate

Extracellular expression

secretion to periplasm

secretion to medium

secretion to medium

secretion to medium

Posttranslational modifications

Protein folding

refolding usually required

refolding may be required

proper folding

proper folding

N-linked glycosylation

none

high mannose

simple, no sialic acid

complex

O-linked glycosylation

no

yes

yes

yes

Phosphorylation

no

yes

yes

yes

Acetylation

no

yes

yes

yes

Acylation

no

yes

yes

yes

gamma-Carboxylation

no

no

no

yes

Gene Expression Systems. Using nature for the art of expression (Fernandez, J.M. & Hoeffler, J.P., eds), Academic Press, San Diego, 1999.

As observed in the table above, Yeast scores above the other types of expression systems in all aspects. This makes it highly acceptable for its use in production of therapeutic proteins. Presence of toxic cell wall pyrogens in E.coli and the presence of potentially harmful oncogenic and viral DNA in mammalian cells, make them least suitable as an expression system. High cell densities of yeast can be achieved when grown on a simple media, and it can be easily manipulated like E.coli. One drawback in using yeast as an expression system is the attachment of high mannose residues. Thus, very few, sialic acid caps are present and as a result, the half life of the protein in serum is affected. In order to solve this proble, scientists have come up with a solution which suggests that if the α-1-

6-mannosyltransferase enzyme (OCH1 gene product) is eliminated, it would help eliminate the problem posed by hypermannosylation. Another solution is the development of high sialic acid residue capped yeast strains by genetic engineering. This helps to increase the protein half life in serum.

Different types of yeast vectors have been developed that allows the constitutive expression of foreign genes. Some types of vectors are as follows:-

Yeast Integrative Vector - These are plasmids that consist of a bacterial vector component and a yeast gene with a selectable marker. They do not possess an origin of replication and a centromere and hence cannot survive in yeast as a free plasmid. The plasmids carry foreign genes into the yeast genome by two different methods. The first method is based on the ntegration of exogenous DNA into the yeast chromosomes at a high frequency by the plasmids that lack an origin for autonomous replication. The second method involves gene replacement by homologous recombination.

Yeast Replicating Vectors - These vectors carry prokaryotic plasmid DNA sequences and a part of Yeast DNA that is derived from the Yeast Integrative vectors. The replicating vectors differ from the integrative vectors in possessing a chromosomal origin of replication. As a result, the transformation efficiency is much higher as compared to the integrative vectors. However, it has been observed that the plasmid is lost as the cell grows. This can be avoided by inserting centromeric sequences.

Yeast Episomal Vectors - These vectors carry prokaryotic sequences, a selectable yeast marker gene, and the entire 2µ plasmid.

Yeast Artificial Chromosomes - These vectors contain an autonomously replicating sequence, a centromeric sequence and a telomeric sequence. Although this vector is highly stable, it is not suitable for expression of foreign proteins in yeast.

Saccharomyces cerevesiae was the first species of yeast that was used for the production of recombinant proteins. Other species of yeast that are used as an expression system include - Pichia pastorus, Hansela polymorpha, Schizosaccaharomyces pombe, Yarrowia lipolytica, Candida utilities, etc. Pichia pastorus and Hansela polymorpha are methylotropic strains which require methanol in their growth medium. Pichia pastorus can be used as an expression system for the production of heterologous proteins. It can be cultured easily, it is easy to manipulate genetically and possesses a secretory pathway that is similar to that which is present in mammalian cells. Glycoproteins can be expressed in Pichia by inducing glycosylation pathways. There are some advantages that favor the selection of Pichia pastorus over Saccharomyces cerevesiae. The first advantage is the presence of a Methanol inducible alcohol Oxidase1 gene (AOX1). This gene is highly regulated and can be used to produce toxic heterologous proteins. It would not have any toxic effect on the host unless the gene is induced. The second advantage is regarding the cell densities. Pichia pastorus can achieve higher cell densities (100g/l dry weight) than Saccharomyces cerevesiae. Schizosaccaharomyces pombe is a prototrophic yeast i.e.they are nutritionally self sufficient and can grow on minimal media. Schizosaccaharomyces pombe reproduces by fission and not via budding as seen in Saccharomyces cerevesiae. It is used in molecular biology studies. It can be used to express eukaryotic genes with introns, a function that is not possible in case of Saccharomyces cerevesiae.

Lady using a tablet
Lady using a tablet

This Essay is

a Student's Work

Lady Using Tablet

This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.

Examples of our work