Expression Systems - for protein production

1. HOSTS FOR GENE CLONING
Dr. Esther Shoba R
Assistant Professor
Department of Life Sciences
Kristu Jayanti College

2. INTRODUCTION
• The development of genetic engineering and cloning has opened
many possibilities of expression and isolation of heterologous
proteins for research purposes.
• Large scale applications such as enzyme, antibody or vaccine
production, the amount of protein required is considerably high.
• In such cases the system in which the protein is expressed must be
easy to culture and maintain, grow rapidly, and produce large
amounts of protein.
• These requirements led to the discovery of protein expression
systems. The various protein expression systems are bacteria, yeast,
insect or mammalian systems.

3. • The following factors determine the type of
expression system used to produce recombinant
proteins:
• time spent in expressing the protein
• ease of handling the expression system
• amount of protein needed
• mass of the protein
• type of post-translational modifications, number of
disulfide bonds
• destination of the expressed protein

4. • The process of expressing a recombinant protein
in an expression system requires the following
information/components.
• identification of the gene that encodes the
protein of interest
• generation of cDNA from the respective mRNA
• selection of suitable expression vector to insert
the gene sequence
• selection of suitable system that can express the
vector
• appropriate screening and scaling up methods

6. Bacterial protein expression systems –
Escherichia coli
• Bacteria act as rapid and simple systems of expressing recombinant
proteins due to the short doubling time.
• The media required to culture them are not expensive and the
methods adapted to scale-up bioproduction are straightforward.
•
• The most widely used host system is E. coli since there is ample
knowledge about its genetics, genome sequence and physiology.
• The genetic manipulation is easy and it also grows to high densities
and is suitable for large-scale fermentations.
• However, the cell wall of E. coli contains toxic pyrogens and the
expressed proteins may have to be extensively tested before use.

8. • Features
• low cost culture methods
• flexible system – can carry plasmids with
multiple promoters, tags and restriction sites
• easy to scale up and produce higher yield of
protein

9. DH5 ALPHA
• DH5 alpha is the most frequently used E. coli strain
for routine cloning applications. In addition to
supporting blue/white screening recA1 and endA1
mutations in DH5a increase insert stability and
improve the quality of plasmid DNA prepared from
minipreps.
• Application:
• Highest transformation efficiency
• General cloning
• Blue-white selection
• Plasmid isolation

10. • The recA1 mutation is a single point mutation that
replaces glycine 160 of the recA polypeptide with
an aspartic acid residue in order to disable the
activity of the recombinases and inactivate
homologous recombination.
• The endA1 mutation inactivates an intracellular
endonuclease to prevent it from degrading the
inserted plasmid.
• High insert stability due to recA1 mutation
• High yield and quality of DNA due to endA
mutation

11. BL 21 HOST CELLS
• BL21 Competent E. coli is a widely used non-
T7 expression E. coli strain and is suitable for
transformation and protein expression.
• Ideal for Plac, Ptacexpression vectors
• Protease deficient

12. BL21 DE 3pLysS
• BL21(DE3)pLysS is a derivative of BL21 that has the T7
RNA polymerase gene under the control of the lacUV5
promoter.
• This arrangement is on a phage genome, called DE3.
• DE3 is inserted into the chromosome of BL21 to make
BL21(DE3).
• pLysS is a plasmid that contains the T7 lysozyme gene
(LysS).
• The T7 lysozyme binds to T7 RNA polymerase causing
inhibition until induction by the addition of IPTG.
• When IPTG is added, the amount of T7 RNA
polymerase increases and overcomes the inhibition by
LysS.

14. • Protein expression from high-copy number plasmids and
powerful promoters will greatly exceed that of any native
host protein, using up valuable resources in the cell thus
leading to slowed growth.
• Additionally, some protein products may be toxic to the host
when expressed, particularly those that are insoluble, act on
DNA, or are enzymatically active.
• For this reason, recombinant proteins are typically expressed
in E. coli engineered to accomodate high protein loads using
inducible promoter systems

16. • ompT: Strains harboring this mutation are deficient in outer
membrane protease VII, which reduces proteolysis of the expressed
recombinant proteins.
• lon protease: Strains where this is completely deleted (designated
lon or Δlon) similary reduce proteolysis of the expressed proteins.
• hsdSB (rB- mB-): These strains have an inactivated native
restriction/methylation system. This means the strain can neither
restrict nor methylate DNA.
• dcm: Similarly, strains with this mutation are unable to methylate
cytosine within a particular sequence.

17. Yeast protein expression systems – Saccharomyces cerevisiae
• The highly developed genetic system, ease of use, reduced
time input and costs have made S. cerevisiae an attractive
organism for the expression and production of recombinant
proteins.
• Yeasts are able to carry specifically designed plasmids and this
ability is valuable in a recombinant protein expression system.
• The plasmid used consists of restriction sites that can be used
to insert the gene sequence of interest.
• Transformation of yeasts with the plasmid produces the
desired protein and can be appropriately scaled up.

18. • Expression of protein using S. cerevisiae involves the following
steps:
• use of competent E. coli cells to take up DNA sequence of interest
• integration of the DNA into bacterial genome or circularization of
the DNA sequence to exist as a plasmid
• selection of transformed E. coli using a selection marker
(antibiotic)
• expansion of selected E. coli in appropriate culture media, such as
classic LB options
• isolation of DNA or plasmid
• transformation into yeast
• screen the transformants for integration of DNA into yeast
chromosome
• selection and scaling-up of high expressing yeast clones in
appropriate culture media
• isolation and purification of intracellular/secreted proteins

19. • Features
• low cost culture methods
• suitable for both intracellular and secreted
proteins
• provides eukaryotic post-translational
glycosylation of proteins although it results in
high

20. • A yeast commonly used for protein production is Pichia
pastoris. Examples of yeast expression vector in Pichia are the
pPIC series of vectors, and these vectors use the AOX1
promoter which is inducible with methanol.
• The plasmids may contain elements for insertion of foreign
DNA into the yeast genome and signal sequence for the
secretion of expressed protein. Proteins with disulphide
bonds and glycosylation can be efficiently produced in yeast.
• Another yeast used for protein production is Kluyveromyces
lactis and the gene is expressed, driven by a variant of the
strong lactase LAC4 promoter