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Recombinant Protein Expression in Escherichia coli (E.coli): What We Need to

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DOI: 10.2174/1381612824666180131121940

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Current Pharmaceutical Design, 2018, 24, 1-8 1
REVIEW ARTICLE

Recombinant Protein Expression in Escherichia coli (E.coli): What We Need to Know

Seyed Mohammad Gheibi Hayat1, Najmeh Farahani2, Behrouz Golichenari3 and Amir Hosein Sahebkar4,*

1
Student Research Committee, Department of Medical Biotechnology, Faculty of Medicine, Mashhad University of Medical Sciences,
Mashhad, Iran; 2Department of Genetics and Molecular Biology, Isfahan University of Medical Sciences, Isfahan, Iran; 3Department
of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran; 4Biotechnology
Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

Abstract: Background: Host, vector, and culture conditions (including cultivation media) are considered among
the three main elements contributing to a successful production of recombinant proteins. Accordingly, one of the
most common hosts to produce recombinant therapeutic proteins is Escherichia coli.
Methodology: A comprehensive literature review was performed to identify important factors affecting produc-
ARTICLE HISTORY tion of recombinant proteins in Escherichia coli.
Results: Escherichia coli is taken into account as the easiest, quickest, and cheapest host with a fully known
Received: October 30, 2017 genome. Thus, numerous modifications have been carried out on Escherichia coli to optimize it as a good candi-
Accepted: January 27, 2017
date for protein expression and; as a result, several engineered strains of Escherichia coli have been designed. In
general; host strain, vector, and cultivation parameters are recognized as crucial ones determining success of
DOI: recombinant protein expression in Escherichia coli. In this review, the role of host, vector, and culture conditions
10.2174/1381612824666180131121940   along with current pros and cons of different types of these factors leading to success of recombinant protein
expression in Escherichia coli were discussed.
Conclusion: Successful protein expression in Escherichia coli necessitates a broad knowledge about physico-
chemical properties of recombinant proteins, selection among common strains of Escherichia coli and vectors, as
well as factors related to media including time, temperature, and inducer.
Keywords: Escherichia coli, cloning, recombinant protein, vector.

1. INTRODUCTION: HOSTS membrane). BL21 includes only E.coli RNA polymerase, while
Escherichia coli (E.coli) is a bacterium that has been widely DE3 has λDE3 lysogen which contains T7 RNA polymerase gene
employed in industrial biotechnology for a long time and it has also under the control of LacUV5 promoter. Therefore, BL21 can be
remained as one of the most suitable options within majority of only recruited for expressing proteins with E.coli RNA polymerase
gene cloning experiments. In this regard; the easiest, quickest, and promoter (lac, tac, trc, ParaBAD, PrhaBAD, and T5) in the up-
cheapest technique in expression of proteins is the use of E.coli, stream of their genes; while DE3 can be similarly used for express-
that is why this bacterium is still inserted in the list of common ing proteins with T7 promoter within the upstream of their genes
hosts for recombinant DNA [1, 2]. (Fig. 1).
Working with this organism is fast, convenient, and also inex- Both of these strains may also have a leaky expression. To ad-
pensive because of its genetic simplicity (it has only about 4400 dress this problem, 1% glucose can be used in the medium. Moreo-
genes), full knowledge of the genome, rapid growth compared with ver, adding glucose is recommended especially when the protein is
that in other simple organisms (the cell division takes 20 minutes), toxic. It should be noted that IPTG is only used to express proteins
high safety compared with that in other organisms, as well as need with T7 promoter in the upstream of their genes (DE3) [6-9].
for cheap media and simple growth conditions (37°C) [3-5].
1.2. BL21 (DE3) pLysS
As a host for expression, E. coli includes numerous strains.
To solve the problem of leaky expression, a new strain called
Thus, proper selection of expression hosts can lead to protein ex-
BL21 (DE3) pLysS was created. This strain is a derivative of BL21
pression efficiency even though such hosts have their own advan-
(DE3) with the difference that the level of T7 RNA polymerase
tages and disadvantages.
before and after induction is low (Fig. 2). Therefore, it has no leaky
Accordingly, the best hosts should be selected based on the expression and it does not require added glucose [10, 11].
requirements in order to obtain the best results (Table 1). In the
BL31 (DE3) pLys also includes one plasmid containing the
following sections, different types of hosts derived from E.coli were
gene expressing the lysozyme, resulting in decomposition of the
described along with their advantages and disadvantages.
enzyme T7 polymerases before induction. However; the lysozyme
1.1. BL21 and BL21 (DE3) can be affected relatively in low levels and there is no adverse ef-
fect due to the very high expression of T7 polymerases after induc-
Both BL21 and BL21 are from E.coli B strains and have defects tion. Likewise, the given strain is compatible with plasmids con-
in the genes of Lon protease (cytoplasm) and OmpT protease (outer taining ColE1 or pMB1 origin. It should be noted Antibiotic Chlo-
ramphenicol is also used for the cultivation of BL21 (DE3) pLysS.
*Address correspondence to this author at the Department of Medical Bio-
technology, School of Medicine, Mashhad University of Medical Sciences, 1.3. BL21 Star
Mashhad, Iran, P.O. Box: 91779-48564, Iran; Tel: 985118002288; This strain of E.coli has a mutation in the gene rne131. Moreo-
Fax: 985118002287; E-mails: sahebkara@mums.ac.ir; ver, the product of gene rne is an endonuclease enzyme called
amir_saheb2000@yahoo.com

1381-6128/18 $58.00+.00 © 2018 Bentham Science Publishers

2 Current Pharmaceutical Design, 2018, Vol. 24, No. 00 Hayat et al.

Table 1. Common E.coli strains used as expression host.

Bacterial Strain Features Benefit Growth Condition Company

BL21(DE3) *Has DE3 lysogen that expresses T7 RNA Suitable for the expression of nontoxic 1 % glucose in the Novagen
polymerase genes medium
*Deficient in lon and ompT proteases
*Induced by IPTG

BL21(DE3) *Has DE3 lysogen that expresses T7 RNA *Prevents leaky expression Chloramphenicol 34 Novagen
pLysS polymerase *Suitable for the expression of toxic genes µg/m
*Has T7 lysozyme to decompose the enzyme
T7 polymerases before induction

BL21 Star Mutation in the gene rne131, so mRNA has Novagen

more stability

Lemo21(DE3) *Contains features of BL21(DE3) Suitable for the expression of challenging L-Rhamnose 0–2,000 NEB
*Tunable expression of difficult clones by protein including: toxic proteins, mem- µM
varying the level of lysozyme (lysY) brane proteins, and low soluble ones

Tuner (DE3) Has lac permease (lacY) mutation *Allows uniform entry of IPTG into all None Novagen
cells in the population.
*Suitable for toxic and insoluble proteins

Origami Has mutation in trxB and gor genes Enhances disulfide bond formation in the Kanamycin 15 µg/mL (Novagen,
cytoplasm Tetracycline 12.5 2006–2007)
µg/mL

SHuffle *Expresses disulfide bond isomerase DsbC *Promotes the correct folding of mis- NEB
*Deficient in proteases Lon and Omp oxidized proteins
*resistance to phage T1 (fhuA2)

Rosetta *BL21 lacZY (Tuner) derivatives Suitable for the expression heterogeneous Chloramphenicol 34 Novagen
*Has additional copies of genes encoding the proteins µg/mL
tRNAs for rare codons AUA, AGG, AGA,
CUA, CCC, GGA

Rosettagami Derived from Origami and also has tRNAs for Novagen
(DE3) rare codons

C41(DE3) and Mutant strains of BL21(DE3) that prevents suitable for the expression of toxic and/or Lucigen
C43(DE3) cell death associated with the expression of membrane proteins from all classes of
toxic recombinant proteins organisms

RNase E with a length of 1061 amino acids. In this respect, studies brane proteins that are in need of secretory systems such as the Sec
have shown that the N-terminal of the enzyme (1-584) can play a translocase or Tat translocase. If the expressed protein is more than
role in rRNA maturation and cell growth, and the C-terminal (585- the capacity of the secretory system, it may appear as an inclusion
1061) has such a role in mRNA degradation. As mentioned earlier; body in cells.
BL21 Star strain in the gene rne has mutation, so the product of this As well, Lemo21 (DE3) allows an adjustable expression of
gene is truncated RNase E without C-terminal domain (Fig.1). difficult proteins if the expression is unregulated which can be ob-
Thus, mRNA in the BL21 Star strains shows higher stability and served by making changes in the levels of natural inhibitor of T7
consequently the efficiency of protein expression increases for het- RNA polymerase and lysozyme (lysY). The addition of L-rhamnose
erologous genes. However, it should be noted that the given strain (between 0 and 2000 µM) into the expression culture can also lead
is not a suitable option for the expression of proteins that are toxic to a modulated level of the lysozyme. The isolation also acts similar
to the host [12-15]. to a pLysS containing strain when no rhamnose is available for
Lemo21 (DE3). Thus, the desired protein expression is regulated by
1.4. Lemo21 (DE3) optional addition of rhamnose (Fig. 3). Tuning the level of expres-
The tuning of T7 expression can have effects on the formation sion probably results in more soluble and proper folded protein for
of inclusion body. In many cases, protein is expressed in a natively proteins that are hard to solve. So, Lemo21 (DE3) is suitable for the
and properly folded form once protein expression is low, but in- expression of some proteins including toxic proteins, membrane
creased expression in a large amount can cause inclusion body. In proteins, and low soluble ones [16-19].
particular, this issue is of utmost importance for secretory or mem-
Recombinant Protein Expression in Escherichia coli (E. coli) Current Pharmaceutical Design, 2018, Vol. 24, No. 00 3

Fig. (1). Schematic view of four types of E.coli strains (BL21 Star, Rosetta, SHuffle and Origami) used in recombinant protein production.

Fig. (2). BL21 (DE3) pLysS has a plasmid containing the gene expressing the lysozyme; resulting in decomposition of the enzyme T7 polymerases before
induction therefore the leaky expression can be prevented.

1.5. Tuner (DE3) 1.6. Origami and SHuffle

An alternative to Lemo21 (DE3) is to use tuner (DE3). These Since appropriate folding of protein needs disulphide bond, the
cells can have an adjustable level of protein expression due to the use of E.coli strain ‘Novagene’ is recommended. The oxidizing
presence of lacZY deletion mutants. In this regard, IPTG can uni- E.coli strain, Origami strain, has mutation in thioredoxin reductase
formly penetrate into all the cells of a population because of muta- (trxB) and glutathione reductase (gor) genes leading to the forma-
tion in the lac permease (lacY) and then create a concentration- tion of disulphide bond in cytoplasm. To express putative disul-
dependent and homogeneous level of induction. It should be noted phide bond-forming protein, ‘SHuffle’ E.coli strains from ‘NEB’
that the regulation of expression is possible from very low to high are better than ‘Origami’ ones. In addition to trxB and gor muta-
levels by adjusting the concentration of IPTG. As mentioned previ- tions, SHuffle strains express DsbC in the cytoplasm which can
ously, the activity and solubility of difficult target proteins resulting direct correct disulfide bond formation and act as a general chaper-
from lower-level expression can be increased [20, 21]. one for protein folding (Fig.1) [22-24].
4 Current Pharmaceutical Design, 2018, Vol. 24, No. 00 Hayat et al.

Fig. (3). In Lemo21 (DE3), T7 RNA polymerase activity can be modulated by T7 lysozyme; on the other hand, the lysozyme level can be modulated by the
concentration of L-rhamnose (between 0 and 2000 µM).

Table 2. Differences between BL21-CodonPlus (DE3)-RIL, BL21-CodonPlus (DE3)-RP and BL21-CodonPlus (DE3)-RIPL cells are
summarized in Table 3.

Host Strain tRNA Gene Antibiotic Resistance

BL21-CodonPlus (DE3)-RIL argU(AGA,AGG), ileY(AUA), leuW(CUA) Camr

BL21-CodonPlus (DE3)-RP argU(AGA, AGG), proL(CCC) Camr

BL21-CodonPlus (DE3)-RIPL argU(AGA,AGG), ileY(AUA), proL(CCC), leuW(CUA) Camr Strep/Specr

1.7. Rosetta (BL21 CodonPlus) tion of heterologous proteins from organisms with GC-rich ge-
The scarcity of special tRNAs in E.coli can largely restrict effi- nomes are also found in the CodonPlus-RP strains.
cient production of heterologous proteins. These tRNAs are avail- Lots of argU, ileY, leuW and proL tRNA genes are similarly
able in high quantities in the organisms from which the heterolo- available in the cells of BL21-CodonPlus (DE3)-RIPL. Further-
gous proteins are derived. The pool of rare tRNAs might be emp- more; the expression of heterologous proteins from organisms with
tied due to high-level expression of heterologous proteins. In this either AT- or GC-rich genomes can occur in this strain [25-27].
respect, additional copies of genes encoding the tRNAs can be Differences between BL21-CodonPlus (DE3)-RIL, BL21-
achieved by BL21-CodonPlus strains. High-level expression of CodonPlus (DE3)-RP and BL21-CodonPlus (DE3)-RIPL cells are
many heterologous recombinant genes in BL21-CodonPlus cells is summarized in Table 3.
also seen due to the high amount of tRNAs which is expressed at
low levels in conventional BL21 strains (Fig. 1). 1.8. C41 (DE3) and C43 (DE3)
Numerous copies of the argU, ileY, and leuW tRNA genes are The mutant strains of C41 (DE3) and C43 (DE3) were designed
found in the cells of BL21-CodonPlus-RIL and BL21-CodonPlus by Miroux and Walker. These mutant strains of E.coli BL21 (DE3)
(DE3)-RIL. The tRNAs detecting arginine codons AGA and AGG, can prevent cell death and plasmid instability associated with the
the isoleucine codon AUA, and the leucine codon CUA are encoded expression of toxic recombinant proteins. These strains are also
by argU, ileY, and leuW tRNA genes; respectively. It should be effective in expressing toxic proteins from all classes of organisms
noted that the tRNAs limiting the translation of heterologous pro- including eubacteria, yeasts, plants, viruses, and mammals. Moreo-
teins from organisms with AT-rich genomes are found in the ver, the given strains have one or lots of mutations which put off s
CodonPlusRIL strains. cell death associated with the expression of toxic proteins [28, 29].
The large quantities of the argU gene (encoding tRNAs for 1.9. Plasmid Vectors
arginine codons AGA and AGG) and proL gene (encoding tRNAs
for proline codon CCC) are available in BL21-CodonPlus-RP and A plasmid is a small, circular, and double-stranded DNA frag-
BL21-CodonPlus (DE3)-RP cells. The tRNAs limiting the transla- ment that can be managed to replicate automatically within the host
Recombinant Protein Expression in Escherichia coli (E. coli) Current Pharmaceutical Design, 2018, Vol. 24, No. 00 5

Table 3. Protein expression troubleshooting in E.coli.

Trouble Reasons Solution

Vector Host Strain Growth Conditions

Incorrect vector Confirms vector by

construction sequencing

Rare codons Codon optimization Uses Rosetta or Codon reduced induction temperature
Plus
No/Low protein
Protein toxicity *Uses promoters with *Uses strains *Start of induction at high OD
expression
tighter regulation pLysS/pLysE or * Shortened induction time
*Lower plasmid copy C41 or C43 * Added glucose when using
number expression vectors containing
lac-based promoters
*Use of defined media with glucose
as a source of carbon

Incorrect *Adds fusion partners Uses Origami or SHuffle *Lower inducer concentration
disulfide bond including thioredoxin, *Lower induction temperature
formation DsbA, and DsbC
*Clones in a vector
containing secretion
signal to cell periplasm

Incorrect folding *Uses a solubilizing Uses strains with *Supplemented media with
partner cold-adapted chaperones chemical chaperones and
*Co-expresses with cofactors
Protein aggrega- molecular chaperones *Removed inducer and added fresh
tion
media
*Lower inducer concentration
*Lower temperature

Proteins with *Adds fusion tags Uses membrane rich *Lower induction temperature
high including GST, MBP, strains (C41/C43) *Shortened induction time
hydrophobicity SUMO, etc. *Growth in poor medium
or *Added heat shock chaperones
transmembrane
domains

Protein Replaces specific protease Uses low protease strains *Induced at high OD
Truncated pro- degradation sites *Induced at low temperature
tein *Shortened induction time
*Use of protease inhibitors when breaking cells

cells and it is also the most frequently used vector within the re- marker), replication origin (Ori), and multiple cloning site (MCS)
combinant DNA approach in E.coli. The purpose of genetic engi- [30, 31].
neering has been the optimization of these kinds of plasmids as Based on their dependence on host proteins, various plasmids
potent vectors in gene cloning. One of the efforts made in this do- have been divided into two categories: stringent plasmids and re-
main is that the length of the plasmids has been shortened to make laxed plasmids. The first category contains plasmids requiring pro-
them easier to work with. Nowadays, the length of many plasmid tein synthesis machinery of the cell, and the second one includes
vectors is only about 3kb that means they are much shorter com- plasmids that do not need this machinery but demand cellular pro-
pared to naturally occurring E.coli plasmids. Likewise, they are a teins previously synthesized in the host cells. It should be noted that
bit more than nucleotides essentials used in gene cloning in most the use of chloramphenicol disrupting protein synthesis of the host
plasmid vectors; for example, antibiotic-resistance gene (selectable
6 Current Pharmaceutical Design, 2018, Vol. 24, No. 00 Hayat et al.

cell does not inhibit the replication of plasmids in the second cate- 1.14. Medium
gory [32, 33]. Given that the increased expression of chaperones helps with
In the following sections, different types of vectors used in correcting the folding of proteins, some chemicals can be used to
genetic engineering were described in order to choose the best vec- induce the chaperones in the host. The expressed proteins can also
tors suitable for cloning purposes and meeting the given needs. reach to more stable status via some small chemicals or ligands. For
instance; the use of ethanol, benzyl alcohol, osmolytes and/or ther-
1.10. pET Vector mal shock and changes in the buffering power can be some of the
Novagen Company (Co.) introduced the pET vector that is factors increasing the expression of the proteins. In addition, these
nowadays considered as the first choice in loads of cloning and factors can boost the expression of proteins in the soluble state and
protein expression projects. The given vector has a high expression also elevate the stability of proteins during the purification process
due to its potent promoter and it is able to simplify protein purifica- [4, 5, 19, 47, 48].
tion using Ni-NTA through adding His-tag to the protein.
2. METHODS
Moreover, there are two strategies to add the His-tag: in C-ter
or in N-ter. Proteins can also be overexpressed without any tag 2.1. Length of Induction
because, in some cases, the His-tag may alter enzymatic properties
Induction is better to be performed when the cells are at the
of the target protein and six consecutive histidines in protein can
maximum concentration. Therefore, cell density is one of the fac-
reduce solubility; thus the given company produced different ver-
tors affecting protein expression. As a result, the best time for in-
sions of this vector termed pET-43 and pET-44. The given vectors
duction is the early mid-log phase though some studies have indi-
can add the NusA (N-utilization substance A) to the N-terminal end
cated that the given induction can be also carried out in mid-log
of the protein and they consequently enhance the solubility of pro-
phase or stationary one. Therefore the best optical density (OD600)
teins. In addition, the pET-Trx plasmid can add thioredoxin to pro-
for induction is between 0.6-0.8 [4, 5, 19, 47-49].
teins that can also increase the protein solubility. PET-39b and
pET-40b plasmids can respectively add DsbA and DsbC fusions to 2.2. Temperature and Duration of Induction
the recombinant protein used to express proteins with disulfide
bond. As well, 208-amino acid DsbA helps with forming a disulfide The duration and temperature of induction are two of the most
bond and 236-amino acid DsbC facilitates the isomerization of important factors affecting expression and/or solubility.
disulfide bond [11, 34-37]. In this respect, decreasing the post-induction temperature can
reduce the protein synthesis rate and consequently prevent the for-
1.11. pHAT Vector mation of inclusion bodies. This approach has been documented to
Given that the use of six consecutive histidines as purification be effective for some of the complicated proteins. Thus, the most
tags can strongly lower protein solubility, Clontech Co. introduced commonly used temperatures for inducing poorly soluble proteins
a vector named pHAT containing 19-amino acid tag. Six out of are 18°, 24° and 30°C. Although cell growth can be increased at
these 19 amino acids are histidines, but these histidines are of non- high temperatures, it can be harmful for protein expression since the
adjacent type; therefore, their overall charge is lower compared higher growth rate can cause a higher possibility of plasmid loss.
with consecutive histidines, so the solubility of proteins with pHAT This can be significant if recombinant proteins are overexpressed in
fusion is higher [38, 39]. continuous cultures.
Generally, aggregation reaction is preferred at higher tempera-
1.12. pMAL and pGEX tures because the hydrophobic interactions are firmly dependent on
In some occasions, proteins with His-tag are not expressed well temperature. Furthermore; low levels of cell division, transcription
and they may form the inclusion body. In this case, the target gene and translation, as well as decreased protein aggregation are caused
can be cloned in vectors such as pGEX or pMAL in order to in- by slow cell processes at the lower temperatures. In this regard,
crease solubility. In this regard, the pGEX vector adds Glutathione lower expression temperature reduces the degradation of proteolyti-
S-transferases (GST) to the obtained protein. This tag with a weight cally sensitive proteins because most proteases at lower tempera-
of 25 kDa can lead to increased protein solubility. The pMAL vec- tures are less active. Therefore, the solubility of proteins can be
tor also adds Maltose Binding Protein (MBP) to the obtained pro- increased by lowering the temperature (with reducing the IPTG
tein. The weight of this tag is 44 kDa. New England Biolabs Co. concentration) and increasing induction duration. Nevertheless, the
provides both of these vectors. The high weight of tags can be a optimum combination of induction duration and post-induction
disadvantage for these vectors. Therefore, this tag can affect the temperature are still obtained by trial and error [4, 5, 19, 47-49].
structure or function of the protein when it is not removed. In addi-
tion, a protease cleavage site has been considered in the vectors to 2.3. Inducer Concentration
remove the fused tag from the protein so that no additional amino Low concentrations of inducer can lead to insufficient induction
acids remain in the protein. Thrombin, SUMO protease, Factor Xa, of the protein expression and thereby lower efficiency. Moreover;
and Enterokinase are documented as the most commonly proteases in addition to having high costs, the high concentration of inducer
used to remove a protein tag [40-44]. can kill cells and prevent protein expression in them due to its toxic
effects on cells. Therefore, regulation of inducer concentration is of
1.13. pSUMO Vector utmost importance and it is better to be a little higher than critical
SUMO tag is added to the end of proteins that have their genes one. According to several studies, it has been demonstrated that
cloned in pSUMO vector. Accordingly, this tag can act as a chaper- samples with IPTG concentrations between 0 and 1 mM cannot
onin and facilitate folding; it can also increase the solubility of pro- have toxic effects on the growth of E.coli. In addition, the use of
teins. LifeSensors Incorporation (Inc.) similarly constructs a variety lower concentration of inducer (e.g., 0.05 mM for IPTG) helps with
of pSUMO vectors that can act as a shuttle vector (E.coli/yeast and expressing the protein in a soluble form, and the use of higher con-
E.coli/mammalian cell). The markers of these vectors are ampicillin centration of the inducer can reduce protein solubility. Finally, it is
and kanamycin. The advantage of these vectors is that the obtained important to note that all the three factors of vector, host, and pro-
protein will not have any additional amino acids after using SUMO tein need to be considered in order to determine the appropriate
protease [45, 46]. concentration of inducer [4, 5, 19, 47-49].
Recombinant Protein Expression in Escherichia coli (E. coli) Current Pharmaceutical Design, 2018, Vol. 24, No. 00 7

2.4. Troubleshooting the purification and extraction phases was assessed using one of the
There are lots of factors disrupting the expression of host pro- fusions.
teins. The sequence at translation initiation region (TIR), the codon One of the desirable cells used in the field of recombinant ex-
usage, and the proper formation of disulfide bonds are of the pivotal pression as a microbial cell factory has always been E.coli. E.coli
factors affecting the expression of protein. The expression of the can be considered as an appropriate host for the expression of
gene can be also strongly affected due to uncommon codons for globe-like proteins from prokaryotes and eukaryotes as well as the
E.coli. Besides, there is a possibility that target genes have codons more stably folded ones. The use of this prokaryote as a precious
at low abundance in this host because of the heterologous feature of means to produce recombinant proteins either in basic research or
the target protein which can result in premature translation termina- in commercial purposes offers many advantages. All of the above-
tion, growth arrest, and decreased protein production yield. Addi- mentioned items in this article including vector, media condition,
tionally, there are two methods to address this issue including the and host can influence the expression of proteins in E.coli. How-
use of de novo gene synthesis to substitute the rare codons in the ever, an expression system which perfectly works with all recombi-
gene sequence (a software was designed to assess the codon context nant proteins cannot be found and it is necessary to obtain an opti-
or codon pair usage in addition to codon frequency), and secondly mum level of stability and high-level synthesis in each case using
the expression of targeted gene in E.coli supplemented by tRNAs at experimental changes in various factors because each protein can
a low level, as mentioned earlier. pose a new challenge. It can be concluded that an appropriate com-
Moreover, it has been indicated that the protein expression lev- bination of the equipment of an extensive genetic toolbox can play
els can be strongly affected by the sequence at the 5′ of the gene a major role in successful preparation of the recombinant proteins
because the translation can be prevented by the formation of secon- in E.coli. The most important factors in this domain are illustrated
dary structures in the mRNA using the ribosome complex. Accord- in Table 3, which should be considered as significant items [5, 48,
ingly, protein expression can be greatly influenced by these se- 50-53].
quences in a direct manner after the start of codon up to the position
CONCLUSION
+25.
Although much progress has been made in the field of heterolo-
Meanwhile, the eukaryotic cells have much longer mRNA half-
gous protein expression in E.coli, expressing a protein with optimal
life compared with bacteria. The mRNA stability has demonstrated
solubility and appropriate structural and functional properties is still
to be increased by the mutation in the gene for RNaseE coding.
a problematic issue. There are numerous strategies to meet these
Invitrogen Co. has presented a strain with the commercial name of
objectives as reviewed in the above sections. In general; host strain,
BL21 Star derived from BL21 which contains respective mutation.
vector, and cultivation parameters are known as crucial parameters
It should be noted that a covalent disulfide bond occurs between determining the success of recombinant protein expression in
two sulfur atoms of two cysteine residues. It is a major property to E.coli. In this regard, successful protein expression in E.coli ini-
be considered in target gene expression because of its pivotal role in tially necessitates a wide knowledge about physicochemical proper-
stability, folding, and/or target protein functioning. The formation ties of the recombinant protein. In the second step, a rational selec-
of disulfide bonds can be seen in environments such as the bacterial tion must be made among common strains of E.coli and vectors.
periplasm or eukaryotic endoplasmic reticulum which are oxidizing Finally; all factors related to media including time, temperature, and
environments. Thus, the functioning of DsbC system in which di- inducer need to be optimized.
sulfide formation is catalyzed by DsbA is necessary in the forma-
tion of disulfide bonds in the periplasmic of E.coli, whereas iso- CONSENT FOR PUBLICATION
merization of wrongly formed disulfide bonds is catalyzed by the
Not applicable.
DsbC. Nevertheless, the saturated translocation machinery can be
considered as a common disadvantage of periplasmic expression CONFLICT OF INTEREST
which may reduce the ultimate efficiency of the target protein with
its possible toxic effect on the host cell. The authors declare no conflict of interest, financial or other-
These negative effects can be reduced as much as possible by wise.
avoiding the saturation of the translocation machinery through util-
ACKNOWLEDGEMENTS
izing a strain with accurately controlled expression intensity such as
Lemo21 (DE3). Accordingly, engineered E.coli strains with more Declared none.
oxidizing cytoplasm have been developed as substitutes for perip-
lasmic expression to optimize the formation of disulfide bonds. REFERENCES
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glutathione reductase (gor) genes which play a role in maintaining Methods and Applications, 2003: 27-48.
the decreased environment in the cytoplasm and a mutation in the [2] Baneyx F. Recombinant protein expression in Escherichia coli.
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