Expression

Systems & Vectors

Overview of Protein Expression
Protein expression refers to the way in which proteins are synthesized, modified and
regulated in living organisms
Introduction to Protein Expression
Proteins are synthesized and regulated depending upon the functional need in the cell. The
blueprints for proteins are stored in DNA and decoded by highly regulated transcriptional
processes to produce messenger RNA (mRNA). The message coded by an mRNA is then
translated into a protein. Transcription is the transfer of information from DNA to mRNA, and
translation is the synthesis of protein based on a sequence specified by mRNA.
Simple diagram of transcription and translation. This describes the general flow of information from DNA base-pair sequence (gene) to amino acid polypeptide sequence
(protein).
In prokaryotes, the process of transcription and translation occur simultaneously

In eukaryotes, the processes are spatially separated and occur sequentially.
Diagram of transcription and translation. This describes the general flow of information from
DNA base-pair sequence (gene) to amino acid polypeptide sequence (protein).
Protein expression is a subcomponent of gene expression. It consists of the stages after
DNA has been transcribed to messenger RNA (mRNA). The mRNA is then translated
into polypeptide chains, which are ultimately folded into proteins.
Choose expression
vector
Choose
vector/fusion tag.
Prepare
vector/insert
construct.
Transform host and
screen for
transformants
Determine
expression levels.
Optimize expression
conditions.
Soluble protein:
purification under
native conditions
Insoluble conditions:
purification under
denaturing
conditions
Overall strategy for recombinant protein expression and purification.
1) Cloning.
2) Expression.
3) Purification.
VARIOUS EXPRESSION SYSTEMS

Cell free systems

Prokaryotic
E. Coli

Eukaryotic
Yeast cells
Insect cells
Mammalian cells
Escherichia coli: The expression of proteins in E. coli is the easiest, quickest and
cheapest method. There are many commercial and non-commercial expression
vectors available with different N- and C-terminal tags and many different strains
which are optimized for special applications.

Yeast: Yeast is an eukaryotic organism and has some advantages and disadvantages
over E. coli. One of the major advantages is that yeast cultures can be grown to very
high densities, which makes them especially useful for the production of isotope
labeled protein for NMR. The two most used yeast strains are Saccharomyces
cerevisiae and the methylotrophic yeast Pichia pastoris.

Baculovirus infected insect cells: Insect cells are a higher eukaryotic system than
yeast and are able to carry out more complex post-translational modifications than
the other two systems. They also have the best machinery for the folding of
mammalian proteins and, therefore, give you the best chance of obtaining soluble
protein when you want to express a protein of mammalian origin. The
disadvantages of insect cells are the higher costs and the longer duration before
you get protein (usually 2 weeks).
10
Advantages of Insect Cell Expression
Convenient vector systems
High-level expression from polyhedrin promoter
Eukaryotic post-translational processing including spilicing.
Simple cell culture conditions
Easy scale-up ; high biomass production
The Foreign DNA sequences can be very large (> 20 Kb ; ~ 700
amino acids) because of open-ended structure of caspid.
Relatively low cost
Improved folding of large proteins
Toxic products are likely to be expressed ( using expression at late
stage of infection)
Powerful, Convenient Expression Systems
DISADVANTAGES

LONG DOUBLING TIME (~20 H)
~12 TIMES HIGHER COST THAN E.coli