The Molecular Biology Notebook OnlineYou are here: Biotechnology > Gene Cloning > Host Cells
Use of Expression Systems in Research and by the Pharmaceutical Industry
- Bacteria - e.g. Escherichia coli (E.coli), Bacillus subtilis (B. subtilis)
- Yeast
- Cultured insect cells
- Cultured mammalian cells
Expression systems are based on the insertion of a gene into a host
cell for its translation
and expression into protein.
Host cells include :-
The choice of cell type used depends upon the protein to be expressed.
All require DNA to be cloned into the an appropriate vector.
Bacteria
- simple physiology
- short generation times, as bacteria grow and multiply rapidly
- large yields of product - up to 10 % of mass (low cost)
- The expressed proteins often do not fold properly and so are biologically inactive.
- The synthesised protein is often toxic to bacteria preventing the cell cultures from reaching high densities. A solution to this problem is to incorporate an inducible promoter, which may be turned on to transcribe the inserted gene after the culture has been grown
- Lack of enzymes responsible for post-translational modifications (effect on function of proteins), eg if the protein to be expressed is a glycoprotein, there is not apparatus in the bacterium to 'stick on' the necessary sugar residues.
Advantages of bacterial cells
With B. subtilis and some others, it is possible to induce secretion
of a gene product into the surrounding medium. This method is in use in the
pharmaceutical industry in the production of hormones such as insulin and
human growth hormone.
Disadvantages of bacterial cells
Yeasts
- Yeast performs many of the post-translational modifications required for human proteins
- Can be induced to secrete certain proteins into the growth medium for harvesting - e.g. Hepatitis B virus (HBV) vaccine.
- Presence of active proteases that degrade foreign (expressed) proteins, thereby reducing their yield (a solution to this problem is the construction of yeast strains from which the protease genes have been deleted).
Yeasts are simple eukaryotes .
Advantages of yeast cells
Disadvantages of yeast cells
Insect Cells
- High level of expression
- Correct folding
- Post-translational modifications similar to those in mammalian cells
- Cost, though more than for culturing bacteria and yeast, less than for mammalian cells e.g. potential vaccine for AIDS virus produced by expression of one of the HIV glycoproteins with this system
- More difficult to work with
- Expensive
- Slow generation time
- Not suitable for proteins with repetitive sequences
Immortal Cell Lines
Immortal cell lines are derived from cancerous mammalian tumours, and so the cells are able to divide and grow at a much faster rate than normal cells.
Tumour Cell Lines are used to study the function and regulation of mammalian genes
Gene Transfer is achieved by :-
- Microinjection of DNA / RNA
- Diethylaminoethyl-Dextran (DEAE Dextran)
- DNA sticks to positively charged DEAE particules
- Large DNA - containing particles stick to surfaces of cells -
- Endocytosis
- Some DNA gets to nucleus - transcribed into RNA
- Calcium Phosphate Co-precipitation
- DNA is precipitated with calcium phosphate
- precipitate pipetted onto cell monolayer
- ...after several hours...
- cells take up precipitated DNA (naked adenovirus DNA)
- precipitate removed from cells
- incubated in fresh growth medium
But DEAE-dextran is very inefficient for many types of cells so other techniques have been developed, such as the transfection of DNA by calcium phosphate co-precipitation
Divalent cations have been found to promote the uptake of DNA
This has been performed with purified tumour virus DNA and has shown that cancer can be encoded in DNA.
Expression of foreign proteins in insect cells through incorporation of their genes into baculovirus vectors
Advantages of insect cells
Disadvantages
Page written by Dr Jonathan Mullins, from the University of Luton
Tweet
Expression Systems on Wikipedia
Expression Systems at Answers. com
Related Links
-MicroArray related activities at the EBIThese pages are a jumping point to activities at the European Bioinformatics Institute that
New Links
-Running DeCypher from Geneious
There is a plugin to connect Geneious and DeCypher together. This page explains how to set it up, an...
-DeCypher on G1
This is the first instance of DeCypher on a G1 card. The oldest. ...
-Decypher on J1
The latest instance (testing). ...
-Decypher on H1
Intermediate DeCypher instance: requires username and login....
-Laboratory Information Management Systems for DNA Barcoding
Here, we demonstrate an example workflow of a specimen’s entry into the LIMS database to the publis...
