Expression systems

Advances inAnimal and Veterinary Sciences
E
An Overview of Heterologous Expression Host Systems for the
Production of Recombinant Proteins
Abstract | With the advent of recombinant DNA
technology, cloning and expression of numerous
mammalian genes in different systems have been explored
to produce many therapeutics and vaccines for human and
animals in the form of recombinant proteins. But selection
of the suitable expression system depends on productivity,
bioactivity, purpose and physicochemical characteristics
of the protein of interest. However, large scale production
of recombinant proteins is still an art in spite of increased
qualitative and quantitative demand for these proteins.
Researchers are constantly challenged to improve and
optimise the existing expression systems, and also to
develop novel approaches to face the demands of
producing the complex proteins. Here, we concisely
review the most frequently used conventional and
alternative host systems, with their unique features, along
with advantages and disadvantages and their potential
appli- cations for the production of recombinant products.
xpression host systems are employed for the expres-
sion of recombinant proteins both for therapy and
research. The need for novel expression host platforms
increases with the number of gene-targets for various in-
dustrial productions. Platforms already in use, range from
bacteria (Baneyx, 1999), yeast (Cregg et al., 2000; Malys
et al., 2011) and filamentous fungi (Visser Hans, 2011) to
cells of higher eukaryotes (Altmann et al., 1999; Kost and
Condreay, 1999). While choosing among these cells, both
economic and qualitative aspects have to be considered.
Industrial production depends on the use of inexpensive
media components and high anticipated economic yield
from the product. The quality of the product is very es-
sential, especially in medicine where production of human
pharmaceuticals is regulated under strict safety aspects
(Gellissen, 2005). Thus, suitable host system needs to be
DNA (rDNA) technology for cloning and subsequent ex-
pression of particular gene of interest of virus/ microbes in
an appropriate system like bacterial / mammalian / insect
/ yeast / plant expression systems will circumvent the dif-
ficulties associated with the production of large quantities
of vaccine or diagnostic agents (Balamurugan et al., 2006).
Such a bio-engineered protein can be obtained in large
amounts in a pure and native form rDNA technology has
necessary tools to produce desired viral/ bacterial proteins
in a native form. The advent of rDNA technology and
its application in the industry has brought about a rapid
growth of biotechnology companies for the production of
the rDNA products in human and animal healthcare.
PROKARYOTIC EXPRESSION SYSTEMS
selected depending on the purpose. Use of recombinant Bacterial expression system is widely used for the expres-
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Advances inAnimal and Veterinary Sciences
sion of rDNA products. They offer severaladvantages viz.,
high level of recombinant protein expression, rapid cell
multiplication and simple media requirement. However,
there are some limitations such as intracellular accumu-
lation of heterologous proteins, improper folding of the
peptide, lack of post-transcriptional modifications, the po-
tential for product degradation due to trace of proteaseim-
purities and production of endotoxin (Balamurugan et al.,
2006). Prokaryotic platforms are widely used to produce
recombinant proteins as these can be easily manipulated
genetically by well-known methods and cultivated in low
costing medium. Among Gram-negative bacteria, E. coli
is extensively used, while Bacillus subtilisis a well-known
Gram-positive producer of recombinant proteins (Watson
et al., 1992). Among the several challenges protein insol-
ubility and low expression levels (especially mammalian
proteins) or post-translational modifications are some of
the intriguing problems while using these systems (Gel-
lissen, 2005).
Escherichia coli
E. coli is a typical prokaryotic expression system and one
of the most attractive heterologous protein producer. The
expression of proteins in thissystem is the easiest, quickest
and cheapest. To date reformed E. coli is the extensively
used cellular host for foreign protein expression because
of its rapid growth rate which is as short as 20-30 minutes
(Snustad and Simmons, 2010), capacity for continuous
fermentation and relatively low cost. There are many com-
mercial and non-commercial expression vectors available
with different N and C terminal tags and many different
strains are being optimized for specialapplications.
There are also problems related to the use of E. coli as pro-
duction host. These problems can be grouped into two cat-
egories: those that are due to the sequence of the gene of
interest and those that are due to the limitations of E. coli as
a host (Brown et al., 2006). In the first category again there
are three ways in which the nucleotide sequence might
prevent efficient expression of a foreign gene. Firstly, the
foreign gene may contain introns which would be a major
problem since E. coli genes do not contain introns and so
the bacterium does not possess necessary machinery for
removing introns from transcripts. Secondly, the foreign
gene might contain sequences which act as termination
signals in E. coli. These sequences are perfectlyinnocuous
in the normal host cell, but in the bacterium it results in
the premature termination and a loss of gene expression.
Thirdly, a problem with codon bias of the gene making E.
coli not an ideal host for translation. Although all organ-
isms use the same genetic code, each organism has a bias
towards preferred codons. This bias reflects the efficiency
with which the tRNA molecules are able to recognize the
different codons. If a cloned gene contains a high pro-
portion of unfavoured codons, the host cell’s tRNAs may
encounter difficulties in translating the gene, reducing the
amount of protein that is synthesized (Brown et al., 2006).
These problems can be solved, with necessary manipula-
tions although it is time consuming. If the gene contains
introns then its complementary DNA (cDNA) prepared
from the mRNA and so lacking introns might be used as
an alternative. In vitro mutagenesis can be employed to
change the sequences of possible terminators and to re-
place un-favoured codons with those preferred by E. coli.
An alternative with genes that are less than 1 kb in length
is to make an artificial version. This involves synthesizing
a set of overlapping oligonucleotides that are ligated to-
gether, the sequences of the oligonucleotides are designed
to ensure that the resulting gene contains preferred E. coli
codons and terminators are absent (Brown et al., 2006).
In the second category, there are three major limitations
in which E. coli may prevent expression of a foreign gene.
Firstly, E. coli might not process the recombinant proteins
correctly especially with respect to post translationalmod-
ifications like N and O linked glycosylation, fatty acid acy-
lation, phosphorylation and disulfide-bond formation that
are required for proper folding of the secondary, tertiary
and quaternary structures and for the functional character-
istics of the protein of interest. Glycosylation is extremely
uncommon in bacteria and recombinant proteins synthe-
sised in E. coliare neverglycosylated correctly. This can af-
fect the bioactivity, function, structure, solubility, stability,
half-life, protease resistance and compartmentalization of
the functional proteins ( Jung and Williams, 1997). Sec-
ondly, E. coli might not correctly fold the recombinant pro-
teins and is unable to synthesise disulphide bonds present
in mammalian proteins. In such case, protein may become
insoluble and forms an inclusion body within the bacte-
rium (Leonhartsberger, 2006). Although proteins can be
recovered from such state, converting them to correct form
is difficult and more often they remain inactive. Recently
it has been identified that the catalytic domain of a cellu-
lase (Cel-CD) from Bacillus subtilis can be secreted into
the medium from recombinant E. coli in large quantities
without its native signal peptide. It has also been proved
that the N-terminal sequence of the full length Cel-CD
played a crucial role in transportation through both inner
and outer membranes. By subcellular location analysis, it
is identified that the secretion is a two-step process via the
SecB-dependent pathway through the inner membrane
and an unknown pathway through the outer membrane
(Gao et al., 2016). Thirdly, E. coli may degrade the recom-
binant protein. Unlike sequence problems, these problems
can be circumvented by using mutant strains of E.coli.
Accumulation of endotoxins (LPS), pyrogenic to humans
and animals, is yet another problem in the production of
therapeutic proteins in E. coli,besides instability of is plas-
July 2016 |Volume 4 |Issue 4| Page 347
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Expression systems

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