Expression Vector

Contains regulatory elements allowing the expression of any foreign DNA it carries

Allow transcription but not a translation of cloned foreign DNA

Allow both the transcription and translation of cloned DNA and thus facilitate the production of recombinant protein

A promoter site and

A terminator site

Site where RNA polymerase binds

It regulates the rate of transcription

Allows the highest possible rate of gene expression to be achieved

Where transcription of a gene is switched on by the addition of a chemical

Where gene transcription is switched off upon addition of a regulatory chemical

Regulates transcription of lacZ gene coding for beta-galactosidase. It can be induced by isopropylthiogalactoside (IPTG). Fusing the lac promoter sequences to target gene will result in the lactose (or IPTG) dependent expression of that gene. However the lac promoter suffers from a number of problems.

First the lac promoter is fairly weak and therefore cannot drive very high levels of protein production and second the lac genes are transcribed to a significant level in the absence of induction

It regulates transcription of a cluster of genes involved in tryptophan biosynthesis. It is repressed by tryptophan and easily induced by 3-beta-indoleacrylic acid.

A hybrid of trp and lac promoter but is stronger than either of them. It is induced by IPTG.

It is a very strong promoter responsible for transcription of lambda DNA molecule in E. coli. It is repressed by a product of lambda cI gene called lambda depressor. Expression vector with lambdaPL promoter is used with mutant E. coli host that synthesises a temperature sensitive form of the lambda repressor protein. At low temperature (>30 C) this mutant lambda repressor protein is able to repress the lambdaPL promoter; at higher temperature the protein is inactivated resulting in transcription of the cloned gene

The heterologous protein production involves suitable expression system

The heterologous protein production involves suitable expression system

No universal expression system

Heterologous protein is not synthesised by the host cell

All expression system have some advantages as well as some disadvantages that should be considered in selecting which one to use

Despite the development of advanced expression vectors there are still numerous difficulties associated with the production of protein from foreign genes cloned in E. coli

Presence of introns in foreign genes

Presence of terminate signals

Codon bias

Processing of proteins - prokaryotes do not carry out the same kind of post-translational modifications such as glycosylation and phosphorylation as eukaryotes do. This affects a protein’s activity or stability or at least its response to antibodies

Processing of proteins - prokaryotes do not carry out the same kind of post-translational modifications such as glycosylation and phosphorylation as eukaryotes do. This affects a protein’s activity or stability or at least its response to antibodies

In prokaryotic expression systems, most protein products of cloned eukaryotic genes become insoluble aggregates called inclusion bodies due to incorrect folding and are very difficult to recover as functional proteins.

Target proteins expressed in E. coli may be mis-folded for a variety of reasons

Advantages of eukaryotic protein expression systems include very high levels of expression.

Easy to purify using special tags which are induced into the vectors including His, Myc and other tags.

Disadvantages of the systems include the fact that eukaryotic cells do grow slower than prokaryotic cells

Are the products of two or more coding sequences from different genes that have been cloned together and that, after translation form a single polypeptide sequence.

It protects the cloned gene product from attack by host cell proteases.

Cloned gene proteins have been found to be resistant to degradation when they are part of a fusion protein, whereas, when expressed as separate intact proteins they are susceptible to degradation by proteolytic enzymes (proteolysis)

Constructed at the DNA level by ligating together a portion of the coding regions of two or more genes. In its simplest form, a fusion vector system entails the insertion of a target gene or gene segment into the coding region of a cloned host gene. Knowledge of the nucleotide sequences of the various coding segments that are joined at the DNA level is necessary to ensure that ligation produces the correct reading frame.