Chapter 3
Recombinant Protein
Production
o Expression systems (bacterial, yeast, insect,
mammalian)
o Use of viral vectors in recombinant protein production
o Case studies: therapeutic proteins and industrial
enzymes
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.
In prokaryotes, the process of transcription and
translation occur simultaneously. The translation of
mRNA starts even before a mature mRNA transcript is
fully synthesized. This simultaneous transcription and
translation of a gene is termed coupled transcription
and translation.
In eukaryotes, the processes are spatially separated and
occur sequentially with transcription happening in the
nucleus and translation, or protein synthesis, occurring
in the cytoplasm.
Traditional strategies for recombinant protein
expression involve transfecting cells with a DNA
vector that contains the template and then
culturing the cells so that they transcribe and
translate the desired protein. Typically, the cells
are then lysed to extract the expressed protein for
subsequent purification. Both prokaryotic and
eukaryotic in vivo protein expression systems are
widely used. The selection of the system depends
on the type of protein, the requirements for
functional activity and the desired yield. These
expression systems are summarized in the table
below and include mammalian, insect, yeast,
bacterial, algal and cell-free. Each system has
advantages and challenges, and choosing the right
system for the specific application is important for
successful recombinant protein expression. The
following table provides an overview of
recombinant protein expression systems.
A general recombinant protein expression workflow
consists of four steps:
•Cloning/construct design—the construction of
expression vectors to carry the target DNA
fragment or gene of interest (GOI)
•Expression—the delivery of the expression
construct to the chosen host cells, the growth of
those cells, and expression of the recombinant
protein
•Purification—the sample preparation and
purification of the target protein
•Analytics/characterization—the identification
and quantitation of the isolated protein of interest
The choice of an optimal expression system for a
specific protein primarily depends on:
The origin of the target protein—Bacterial
proteins are better expressed in bacterial systems,
while mammalian proteins are better expressed in
mammalian systems. For example, mammalian
cell lines such as Chinese Hamster Ovary (CHO)
and Human Embryonic Kidney (HEK) are the
systems of choice for human protein production.
Required post-translational modifications—
Proteins devoid of complex modifications (e.g.,
glycosylation, alkylation, phosphorylation, or
specific proteolytic processing) are more easily
produced in simpler systems with short turnaround
times such as bacteria and yeast cells. In contrast,
complex proteins with significant modifications
should be produced in either insect or mammalian
cells.
Solubility of the recombinant protein—Some
proteins are not properly folded in bacterial
systems, as a result, they tend to form insoluble
aggregates (or inclusion bodies) that are difficult to
extract. In these circumstances, higher eukaryotic
systems should be preferred for the recombinant
production of the target protein.
Each system has advantages and challenges,
making host choice very important for successful
recombinant protein expression. We’ve compared
in the enclosed table the different host systems for
each critical parameter.
Mammalian protein expression
Mammalian expression systems can be used to
produce proteins transiently or through stable cell
lines, where the expression construct is integrated
into the host genome. While stable cell lines can
be used over several experiments, transient
production can generate large amounts of protein
in one to two weeks. These transient, high-yield
mammalian expression systems utilize suspension
cultures and can produce gram-per-liter yields.
Furthermore, these proteins have more native
folding and post-translational modifications, such
as glycosylation, as compared to other expression
systems. In the example that follows, 3 different
mammalian expression systems were used to
express recombinant proteins.
Mammalian expression systems can be used to
produce mammalian proteins that have the most
native structure and activity due to its
physiologically relevant environment. This results
in high levels of post-translational processing and
functional activity. Mammalian expression systems
are the preferred system for the expression of
mammalian proteins and can be used for the
production of antibodies, complex proteins and
proteins for use in functional cell-based assays.
However, these benefits are coupled with more
demanding culture conditions.
Insect protein expression
Insect cells can be used for high level protein
expression with modifications similar to
mammalian systems. There are several systems
that can be used to produce recombinant
baculovirus, which can then be utilized to express
the protein of interest in insect cells. These
systems can be easily scaled up and adapted to
high-density suspension culture for large-scale
expression of protein that is more functionally
similar to native mammalian protein. Though
yields can be up to 500 mg/L, recombinant
baculovirus production can be time consuming and
culture conditions more challenging than
prokaryotic systems.
Yeast protein expression
Yeast strains are extremely useful for the
expression and analysis of recombinant eukaryotic
proteins and are ideally suited for large-scale
production. Yeast systems also are typically used
for structural and functional research such as
protein interaction studies. These single-celled
eukaryotic organisms are genetically well
characterized and are known to perform many
post-translational modifications. They grow quickly
in defined medium and are easily adapted to
fermentation methods. Yeast systems are also
easier and less expensive to work with than insect
or mammalian cells.
Bacterial expression
Bacterial protein expression systems are popular
because bacteria are easy to culture, grow fast and
produce high yields of recombinant protein.
However, multi-domain eukaryotic proteins
expressed in bacteria often are non-functional
because the cells are not equipped to accomplish
the required post-translational modifications or
molecular folding. Also, many proteins become
insoluble as inclusion bodies that are very difficult
to recover without harsh denaturants and
subsequent cumbersome protein-refolding
procedures. In the example that follows, a bacterial
cell-based system was used to express 8 different
recombinant proteins.