Asian Pac J Trop Biomed 2012; 2(2): 159-162 159

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Asian Pacific Journal of Tropical Biomedicine

journal homepage:www.elsevier.com/locate/apjtb

Document heading doi:10.1016/S2221-1691(11)60213-X 襃 2012 by the Asian Pacific Journal of Tropical Biomedicine. All rights reserved.

Strategies for production of active eukaryotic proteins in bacterial

expression system
Orawan Khow, Sunutcha Suntrarachun*
Queen Saovabha Memorial Institute, Thai Red Cross Society, Bangkok 10330, Thailand

ARTICLE INFO ABSTRACT

Article history: Bacteria have long been the favorite expression system for recombinant protein production.
Received 25 July 2011 However, the flaw of the system is that insoluble and inactive proteins are co-produced due
Received in revised form 20 August 2011
to codon bias, protein folding, phosphorylation, glycosylation, mRNA stability and promoter
Accepted 1 September 2011
strength. Factors are cited and the methods to convert to soluble and active proteins are
Available online 28 February 2012
described, for example a tight control of Escherichia coli milieu, refolding from inclusion body
and through fusion technology.
Keywords:
Recombinant protein
Expression
Escherichia coli

1. Introduction
Most of amino acids have been encoded by more than one
Bacterial expression is the most common expression codon and all available amino acid codons are bias utilized
system employed for the production of recombinant by each organism. Transfer RNA (tRNA) of cells reflects
proteins. The organism, especially Escherichia coli (E. coli), the codon bias of its mRNA. Observation of codon usage in
is easy to manipulate, inexpensive in culturing and fast in E. coli reveals that highly expressed genes exhibit greater
generation of a recombinant protein. However, since it is a codon bias than poorly expressed ones and the frequency
prokaryotic based system, heterologous eukaryotic proteins of synonymous codons used reflects the abundance of their
expressed are not correctly modified, and it can also be cognate tRNAs. These can imply that heterologous genes
difficult to facilitate the secretion of expressed protein with abundant codons, rarely used in E. coli, may not be
in large amounts. Moreover, proteins expressed in large efficiently expressed in E. coli and may lead to translation
amounts might precipitate, forming inclusion bodies and error. C odon bias becomes highly prevalent problems
large complex proteins could be difficult to propagate[1]. when rare codons in the transcripts form clusters such as
doublets or triplets accumulation that is large in quantities.
Translation error arised from rare codon bias includes
2. Factors affecting bacterial expression system mistranslational amino acid substitutions, frameshifting
events or premature translational termination[2-4].
2.1. Codon usage/bias
2.2. Protein folding

*Corresponding author: Sunutcha Suntrarachun, Research and Development, Queen Expression of recombinant proteins in E. coli is mainly
Saovabha Memorial Institute, Thai Red Cross Society, Bangkok 10330, Thailand, 1871 directed to three different locations i.e. the cytoplasm, the
Rama IV Rd., Patumwan, Bangkok 10330, Thailand.
Tel: 66-2-2520161-4 ext 161 periplasm, and the growth medium (through secretion).
Fax: 66-2-2540212 E xpression in the cytoplasm is preferred since the
E-mail: sunutcha@yahoo.com
Foundation Project: This work was financially supported by Queen Saovabha Memorial production yields are usually high. Cytoplasmic folding is
Institute, The Thai Red Cross Socity. often enhanced at low temperatures thus the use of cold-
160 Orawan Khow and Sunutcha Suntrarachun /Asian Pac J Trop Biomed 2012; 2(2): 159-162

inducible promoters may facilitate this process. However, and expensive[2,9-11].

this is often accompanied by misfolding and segregation
into insoluble aggregates known as inclusion bodies.
Aggregation can be reduced to minimum through the control 3. Strategies for improving the expression of active and
of parameters such as temperature, expression rate and host soluble protein
metabolism. Though formation of inclusion body renders
easier protein purification, there is no guarantee that the in 3.1. Tight control of the E. coli cellular milieu
vitro refolding will generate large amounts of biologically
active products. To release recombinant proteins into the Expression of soluble proteins can be regulated through
periplasm and the growth medium, many systems have been many factors that the host cell normally use in controlling of
studied. As such a approach is complicated, the systems toxic protein expression[6,8,12-14].
have not been commercialized[1,2,5].
3.1.1. Modification of E. coli host strain
2.3. Protein phosphorylation and glycosylation The strain or genetic background of host strain is important
for recombinant protein expression. Expression strains
E. coli has limited eukaryotic post-translational machinery should be deficient in harmful proteases, but should stably
function, which is considered as a key disadvantage for maintain the expression plasmid and confer the relevant
producing the eukaryotic phosphoproteins i.e. serine/ genetic elements to the expression system. DE3. E. coli BL21
threonine/tyrosine protein kinases. T o overcome these is an example of the most common host and it has been
obstacles, co-expression of modified mammalian enzymes proven outstanding in application for standard recombinant
such as protein methylases and acetylases and their expression. It can grow efficiently in minimal media as non-
substrates from single or two separate plasmid vectors in the pathogenic bacterium that cannot survive to cause diseases
same E. coli may result in the production of recombinant in host tissues[9,11,15,16].
proteins that closely resemble native eukaryotic proteins[2].
G lycosylation is another complex process of post- 3.1.2. Modification of media composition
translational modification. It is responsible for the formation Production of recombinant protein requires nutrients for
of cellular glycans which are often attached to proteins bacterial growth and there is a limited control on the growth
and lipids. G lycosyltransferase and glycosidases are parameters. This process often leads to changes in substrate
enzymes responsible for glycosylation of many proteins. depletion, pH, and concentration of dissolved oxygen as
Glycoproteins, which are commonly distributed in eukaryotic well as accumulation of inhibitory substances from various
cells, are rarely presented in prokaryotic organisms because metabolic pathways. These changes are not beneficial for
cellular organelles essential for glycosylation are missing in the production of either soluble or correctly folded active
these organisms[4,6-8]. protein. Proper and efficient protein folding might require
specific cofactors in the growth media such as metal ions.
2.4. Stability of mRNA Addition of these essential factors to the culture media could
considerably increase the yield as well as the folding rate of
T he stability of m RNA affects expression rates. T he the soluble proteins[4,17].
average half-life of mRNA in E. coli at 37 曟 ranges from
seconds to the maximum at 20 min and the expression 3.1.3. Expression at lower temperatures
rate depends directly on the inherent stability of mRNA. Protein expression in E. coli growing at low temperature
Degradation of mRNA by RNases can be protected through has shown its success in improving the solubility of proteins
RNA folding, ribosomes and stability modulation by that are difficult to express as soluble proteins. Expression at
polyadenylation. Recombinant expression systems with low temperature conditions leads to the increase of stability
mRNA stability enhancement is commercially available, for and correct folding patterns due to the fact that hydrophobic
example, Invitrogen BL21 star strain, containing a mutation interactions determining inclusion body formation are
in the gene encoding RNaseE[1,8,9]. temperature dependent. Moreover, any expression associated
with toxic phenotype observed at 37 曟 incubation conditions,
2.5. Promoter strength will be suppressed at low temperatures. The increase of
expression and activity of lower temperatures growth is
R ecombinant expression plasmids require strong associated with increased expression of chaperones in E.
transcriptional promoter to enable high-level gene coli. Therefore, growth at a temperature range of (15-23) 曟,
expression. Promoter must be induced using either thermal could also lead to a significant reduction of expressed protein
or chemical means and the most common inducer is the degradation[4,18,19].
sugar molecular isopyl-beta- D -thiogalactopyranoside
( IPTG ) . H owever, IPTG is not suitable for large scale 3.1.4. Co-expression of molecular chaperones
production of human therapeutic proteins because it is toxic Molecular chaperones are proteins adapted to assist
 Orawan Khow and Sunutcha Suntrarachun /Asian Pac J Trop Biomed 2012; 2(2): 159-162
161

de novo protein folding and /or facilitate expressed inclusion body could lead to non-native conformation of
polypeptide’s proper conformation attainment. C o- the expressed protein. This problem could be resolved
expression of molecular chaperone strategy has been by proper refolding procedures of target protein at low
adopted for prevention of inclusion body formation, leading denaturant concentrations. Higher concentration of the
to improving of solubility of the recombinant protein. unfolded protein often leads to decreased refolding yields,
Chaperones are working as a trigger factor assisting in regardless of refolding method. So, it is desirable to keep
recombinant protein refolding. These polypeptides continue the concentration of the initial un-folded protein to a
to attain folding into the native state even after their release minimum level if higher and correct refolding proteins are
from the protein-chaperone complex. M oreover, some expected[1,3].
chaperons could also prevent protein aggregation[2,20-22].
3.3. Active proteins production
3.2. Inclusion body folding
3.3.1. Production of fusion protein
Inclusion bodies are intracellular protein aggregates In order to simplify the expression, solubilization and
which were observed when the target gene is over expressed purification of recombinant proteins, a wide range of protein
in the cytoplasm of E. coli. Formation of inclusion bodies fusion partners have been developed. These fusion proteins
in recombinant expression systems occurs as a result of usually include a partner or “tag” which may be linked to
erroneous equilibrium between in vitro protein solubilization the target protein by a recognition site-specific protease.
and aggregation and might lead to unfavorable protein Most fusion partners are exploited for the purpose of specific
folding[23-25]. affinity purification. Several different tags are commercially
available and offer additional advantages such as protection
3.2.1. Refolding/resolubilization of E. coli inclusion body of partner protein from intracellular proteolysis, enhanced
proteins solubility and they can also be used as specific expression
Recombinant proteins expressed as inclusion bodies in reporters. The most popular affinity tags are poly-histidine
E. coli have been widely used for the commercial product (His6) tags, which are compatible with immobilized metal
of therapeutic proteins. The major drawbacks during the affinity chromatography ( IMAC ) and the glutathione
refolding of inclusion body proteins into more efficient, S-transferase (GST) tag for purification through glutathione
soluble and correct folded product are reducing of recovery. based resins. GST, 27 kDa, can be prohibitive due to the slow
Other than that the requirement for optimization of refolding binding kinetics of GST to glutathione-sepharose resin and
conditions for each target protein and the resolubilization lead to loading of cell extracts extremely time consuming,
procedure could possibly affect the activity of refolded especially when large cell culture volumes are being
protein. Therefore, the production of soluble recombinant processed. Poly-histidine tags, on the other hand, are small
protein remains a preferable alternative than the in vitro and do not, in most cases, affect the folding of the attached
refolding procedures[1,9,23-25]. protein. It also has very strong reversible binding attributes
allowing for rapid single-step purification. Polyhistidine
3.2.2. Isolation/solubilization of inclusion bodies tags can be attached on either the N- or C- termini of
Isolation of inclusion bodies can be done by lysozyme recombinant proteins, but the optimal location depends on
treatment along with EDTA before cell homogenization to the folding and biochemical characteristics of the adjacent
facilitate cell disruption. Inclusion bodies are recovered recombinant protein[14,20,26,27].
by low speed centrifugation of bacterial cells that has
been mechanically disrupted either by ultrasonication 3.3.2. Site-specific protease
or high pressure homogenization. Bacterial cell envelop The recombinant purified protein can be separated from
or outer membrane proteins may co-precipitate with the its fusion partners, such as affinity tags, solubility enhancers
insoluble fractions as the inclusion body impurities. These or expression reporters, by site-specific proteases. These
contaminants can easily be removed by adding detergents enzymes are highly efficient for the cleavage at the inserted
such as Triton X-100 or low concentrations of chaotropic recognition sequence. Selection of specific protease and its
compounds. After removal of the impurities, inclusion bodies optimal cleavage conditions mostly depend upon the amino
are solubilized by various concentrations of chaotropic acid sequences of target protein. Therefore, fusion tags with
agents such as urea or guanidinium hydrochloride. The protease cleavage sequences similar to the one present
latter is more favored due to its better chaotropic properties. in recombinant protein should be avoided. Two serine
Inclusion body proteins that were solubilized under mild proteases, factor Xa and thrombin, are widely used for site-
denatured conditions are better in refolding yields and specific fusion protein cleavage. Enterokinase and tobacco
retaining of biological activities[4,9,23-25]. etch virus protease (TEV) are examples of specific proteases
used in intracellular processing of fusion proteins[9,25].
3.2.3. Refolding of solubilized and unfolded proteins
T he methods normally used for solubilization of
162 Orawan Khow and Sunutcha Suntrarachun /Asian Pac J Trop Biomed 2012; 2(2): 159-162

4. Conclusion recombinant protein expression in Escherichia coli. J Biotechnol

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