Recombinant Protein Expression in Escherichia coli

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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. ARTICLE HISTORY Received: October 30, 2017 Accepted: January 27, 2017 DOI: 10.2174/1381612824666180131121940

Methodology: A comprehensive literature review was performed to identify important factors affecting production of recombinant proteins in Escherichia coli. Results: Escherichia coli is taken into account as the easiest, quickest, and cheapest host with a fully known genome. Thus, numerous modifications have been carried out on Escherichia coli to optimize it as a good candidate 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 recombinant protein expression in Escherichia coli. In this review, the role of host, vector, and culture conditions 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 physicochemical 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 Escherichia coli (E.coli) is a bacterium that has been widely employed in industrial biotechnology for a long time and it has also remained as one of the most suitable options within majority of gene cloning experiments. In this regard; the easiest, quickest, and cheapest technique in expression of proteins is the use of E.coli, that is why this bacterium is still inserted in the list of common hosts for recombinant DNA [1, 2]. Working with this organism is fast, convenient, and also inexpensive because of its genetic simplicity (it has only about 4400 genes), full knowledge of the genome, rapid growth compared with that in other simple organisms (the cell division takes 20 minutes), high safety compared with that in other organisms, as well as need for cheap media and simple growth conditions (37°C) [3-5]. As a host for expression, E. coli includes numerous strains. Thus, proper selection of expression hosts can lead to protein expression efficiency even though such hosts have their own advantages and disadvantages. Accordingly, the best hosts should be selected based on the requirements in order to obtain the best results (Table 1). In the following sections, different types of hosts derived from E.coli were described along with their advantages and disadvantages. 1.1. BL21 and BL21 (DE3) Both BL21 and BL21 are from E.coli B strains and have defects in the genes of Lon protease (cytoplasm) and OmpT protease (outer *Address correspondence to this author at the Department of Medical Biotechnology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran, P.O. Box: 91779-48564, Iran; Tel: 985118002288; Fax: 985118002287; E-mails: [email protected]; [email protected] 1381-6128/18 $58.00+.00

membrane). BL21 includes only E.coli RNA polymerase, while DE3 has λDE3 lysogen which contains T7 RNA polymerase gene under the control of LacUV5 promoter. Therefore, BL21 can be only recruited for expressing proteins with E.coli RNA polymerase promoter (lac, tac, trc, ParaBAD, PrhaBAD, and T5) in the upstream of their genes; while DE3 can be similarly used for expressing proteins with T7 promoter within the upstream of their genes (Fig. 1). Both of these strains may also have a leaky expression. To address this problem, 1% glucose can be used in the medium. Moreover, adding glucose is recommended especially when the protein is toxic. It should be noted that IPTG is only used to express proteins with T7 promoter in the upstream of their genes (DE3) [6-9]. 1.2. BL21 (DE3) pLysS To solve the problem of leaky expression, a new strain called BL21 (DE3) pLysS was created. This strain is a derivative of BL21 (DE3) with the difference that the level of T7 RNA polymerase before and after induction is low (Fig. 2). Therefore, it has no leaky expression and it does not require added glucose [10, 11]. BL31 (DE3) pLys also includes one plasmid containing the gene expressing the lysozyme, resulting in decomposition of the enzyme T7 polymerases before induction. However; the lysozyme can be affected relatively in low levels and there is no adverse effect due to the very high expression of T7 polymerases after induction. Likewise, the given strain is compatible with plasmids containing ColE1 or pMB1 origin. It should be noted Antibiotic Chloramphenicol is also used for the cultivation of BL21 (DE3) pLysS. 1.3. BL21 Star This strain of E.coli has a mutation in the gene rne131. Moreover, the product of gene rne is an endonuclease enzyme called © 2018 Bentham Science Publishers

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Table 1.

Hayat et al.

Common E.coli strains used as expression host.

Bacterial Strain

Features

Benefit

Growth Condition

Company

BL21(DE3)

*Has DE3 lysogen that expresses T7 RNA polymerase

Suitable for the expression of nontoxic genes

1 % glucose in the medium

Novagen

*Prevents leaky expression

Chloramphenicol 34 µg/m

Novagen

*Deficient in lon and ompT proteases *Induced by IPTG BL21(DE3) pLysS

*Has DE3 lysogen that expresses T7 RNA polymerase

*Suitable for the expression of toxic genes

*Has T7 lysozyme to decompose the enzyme T7 polymerases before induction BL21 Star

Mutation in the gene rne131, so mRNA has more stability

Lemo21(DE3)

*Contains features of BL21(DE3) *Tunable expression of difficult clones by varying the level of lysozyme (lysY)

Tuner (DE3)

Has lac permease (lacY) mutation

Novagen Suitable for the expression of challenging protein including: toxic proteins, membrane proteins, and low soluble ones

L-Rhamnose 0–2,000 µM

NEB

*Allows uniform entry of IPTG into all cells in the population.

None

Novagen

Kanamycin 15 µg/mL

(Novagen, 2006–2007)

*Suitable for toxic and insoluble proteins Origami

SHuffle

Has mutation in trxB and gor genes

*Expresses disulfide bond isomerase DsbC *Deficient in proteases Lon and Omp

Enhances disulfide bond formation in the cytoplasm

Tetracycline 12.5 µg/mL

NEB

*Promotes the correct folding of misoxidized proteins *resistance to phage T1 (fhuA2)

Rosetta

*BL21 lacZY (Tuner) derivatives *Has additional copies of genes encoding the tRNAs for rare codons AUA, AGG, AGA, CUA, CCC, GGA

Rosettagami (DE3)

Derived from Origami and also has tRNAs for rare codons

C41(DE3) and C43(DE3)

Mutant strains of BL21(DE3) that prevents cell death associated with the expression of toxic recombinant proteins

Suitable for the expression heterogeneous proteins

Chloramphenicol 34 µg/mL

Novagen

Novagen suitable for the expression of toxic and/or membrane proteins from all classes of organisms

RNase E with a length of 1061 amino acids. In this respect, studies have shown that the N-terminal of the enzyme (1-584) can play a role in rRNA maturation and cell growth, and the C-terminal (5851061) has such a role in mRNA degradation. As mentioned earlier; BL21 Star strain in the gene rne has mutation, so the product of this gene is truncated RNase E without C-terminal domain (Fig.1). Thus, mRNA in the BL21 Star strains shows higher stability and consequently the efficiency of protein expression increases for heterologous genes. However, it should be noted that the given strain is not a suitable option for the expression of proteins that are toxic to the host [12-15]. 1.4. Lemo21 (DE3) The tuning of T7 expression can have effects on the formation of inclusion body. In many cases, protein is expressed in a natively and properly folded form once protein expression is low, but increased expression in a large amount can cause inclusion body. In particular, this issue is of utmost importance for secretory or mem-

Lucigen

brane proteins that are in need of secretory systems such as the Sec translocase or Tat translocase. If the expressed protein is more than the capacity of the secretory system, it may appear as an inclusion body in cells. As well, Lemo21 (DE3) allows an adjustable expression of difficult proteins if the expression is unregulated which can be observed by making changes in the levels of natural inhibitor of T7 RNA polymerase and lysozyme (lysY). The addition of L-rhamnose (between 0 and 2000 µM) into the expression culture can also lead to a modulated level of the lysozyme. The isolation also acts similar to a pLysS containing strain when no rhamnose is available for Lemo21 (DE3). Thus, the desired protein expression is regulated by optional addition of rhamnose (Fig. 3). Tuning the level of expression probably results in more soluble and proper folded protein for proteins that are hard to solve. So, Lemo21 (DE3) is suitable for the expression of some proteins including toxic proteins, membrane proteins, and low soluble ones [16-19].

Recombinant Protein Expression in Escherichia coli (E. coli)

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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) An alternative to Lemo21 (DE3) is to use tuner (DE3). These cells can have an adjustable level of protein expression due to the presence of lacZY deletion mutants. In this regard, IPTG can uniformly penetrate into all the cells of a population because of mutation in the lac permease (lacY) and then create a concentrationdependent and homogeneous level of induction. It should be noted that the regulation of expression is possible from very low to high levels by adjusting the concentration of IPTG. As mentioned previously, the activity and solubility of difficult target proteins resulting from lower-level expression can be increased [20, 21].

1.6. Origami and SHuffle Since appropriate folding of protein needs disulphide bond, the use of E.coli strain ‘Novagene’ is recommended. The oxidizing E.coli strain, Origami strain, has mutation in thioredoxin reductase (trxB) and glutathione reductase (gor) genes leading to the formation of disulphide bond in cytoplasm. To express putative disulphide bond-forming protein, ‘SHuffle’ E.coli strains from ‘NEB’ are better than ‘Origami’ ones. In addition to trxB and gor mutations, SHuffle strains express DsbC in the cytoplasm which can direct correct disulfide bond formation and act as a general chaperone for protein folding (Fig.1) [22-24].


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) The scarcity of special tRNAs in E.coli can largely restrict efficient production of heterologous proteins. These tRNAs are available in high quantities in the organisms from which the heterologous proteins are derived. The pool of rare tRNAs might be emptied due to high-level expression of heterologous proteins. In this respect, additional copies of genes encoding the tRNAs can be achieved by BL21-CodonPlus strains. High-level expression of many heterologous recombinant genes in BL21-CodonPlus cells is also seen due to the high amount of tRNAs which is expressed at low levels in conventional BL21 strains (Fig. 1). Numerous copies of the argU, ileY, and leuW tRNA genes are found in the cells of BL21-CodonPlus-RIL and BL21-CodonPlus (DE3)-RIL. The tRNAs detecting arginine codons AGA and AGG, the isoleucine codon AUA, and the leucine codon CUA are encoded by argU, ileY, and leuW tRNA genes; respectively. It should be noted that the tRNAs limiting the translation of heterologous proteins from organisms with AT-rich genomes are found in the CodonPlusRIL strains. The large quantities of the argU gene (encoding tRNAs for arginine codons AGA and AGG) and proL gene (encoding tRNAs for proline codon CCC) are available in BL21-CodonPlus-RP and BL21-CodonPlus (DE3)-RP cells. The tRNAs limiting the transla-

tion of heterologous proteins from organisms with GC-rich genomes are also found in the CodonPlus-RP strains. Lots of argU, ileY, leuW and proL tRNA genes are similarly available in the cells of BL21-CodonPlus (DE3)-RIPL. Furthermore; the expression of heterologous proteins from organisms with either AT- or GC-rich genomes can occur in this strain [25-27]. Differences between BL21-CodonPlus (DE3)-RIL, BL21CodonPlus (DE3)-RP and BL21-CodonPlus (DE3)-RIPL cells are summarized in Table 3. 1.8. C41 (DE3) and C43 (DE3) The mutant strains of C41 (DE3) and C43 (DE3) were designed by Miroux and Walker. These mutant strains of E.coli BL21 (DE3) can prevent cell death and plasmid instability associated with the expression of toxic recombinant proteins. These strains are also effective in expressing toxic proteins from all classes of organisms including eubacteria, yeasts, plants, viruses, and mammals. Moreover, the given strains have one or lots of mutations which put off s cell death associated with the expression of toxic proteins [28, 29]. 1.9. Plasmid Vectors A plasmid is a small, circular, and double-stranded DNA fragment that can be managed to replicate automatically within the host


Table 3.


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Protein expression troubleshooting in E.coli.

Trouble

Reasons

Solution Vector

Incorrect vector construction

Confirms vector by sequencing

Rare codons

Codon optimization

Host Strain

Growth Conditions

Uses Rosetta or Codon

reduced induction temperature

Plus No/Low protein expression

Protein toxicity

*Uses promoters with tighter regulation

*Uses strains pLysS/pLysE or

*Lower plasmid copy

C41 or C43

number

*Start of induction at high OD * Shortened induction time * Added glucose when using expression vectors containing lac-based promoters *Use of defined media with glucose as a source of carbon

Incorrect

*Adds fusion partners

disulfide bond

including thioredoxin,

formation

DsbA, and DsbC

Uses Origami or SHuffle

*Lower inducer concentration *Lower induction temperature

*Clones in a vector containing secretion signal to cell periplasm Incorrect folding

Protein aggregation

*Uses a solubilizing

Uses strains with

*Supplemented media with

partner

cold-adapted chaperones

chemical chaperones and

*Co-expresses with

cofactors

molecular chaperones

*Removed inducer and added fresh 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

Truncated protein

Protein

Replaces specific protease

degradation

sites

Uses low protease strains

*Induced at high OD *Induced at low temperature *Shortened induction time *Use of protease inhibitors when breaking cells

cells and it is also the most frequently used vector within the recombinant DNA approach in E.coli. The purpose of genetic engineering has been the optimization of these kinds of plasmids as potent vectors in gene cloning. One of the efforts made in this domain is that the length of the plasmids has been shortened to make them easier to work with. Nowadays, the length of many plasmid vectors is only about 3kb that means they are much shorter compared to naturally occurring E.coli plasmids. Likewise, they are a bit more than nucleotides essentials used in gene cloning in most plasmid vectors; for example, antibiotic-resistance gene (selectable

marker), replication origin (Ori), and multiple cloning site (MCS) [30, 31]. Based on their dependence on host proteins, various plasmids have been divided into two categories: stringent plasmids and relaxed plasmids. The first category contains plasmids requiring protein synthesis machinery of the cell, and the second one includes plasmids that do not need this machinery but demand cellular proteins previously synthesized in the host cells. It should be noted that the use of chloramphenicol disrupting protein synthesis of the host


cell does not inhibit the replication of plasmids in the second category [32, 33]. In the following sections, different types of vectors used in genetic engineering were described in order to choose the best vectors suitable for cloning purposes and meeting the given needs. 1.10. pET Vector Novagen Company (Co.) introduced the pET vector that is nowadays considered as the first choice in loads of cloning and protein expression projects. The given vector has a high expression due to its potent promoter and it is able to simplify protein purification using Ni-NTA through adding His-tag to the protein. 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 because, in some cases, the His-tag may alter enzymatic properties of the target protein and six consecutive histidines in protein can reduce solubility; thus the given company produced different versions of this vector termed pET-43 and pET-44. The given vectors can add the NusA (N-utilization substance A) to the N-terminal end of the protein and they consequently enhance the solubility of proteins. In addition, the pET-Trx plasmid can add thioredoxin to proteins that can also increase the protein solubility. PET-39b and pET-40b plasmids can respectively add DsbA and DsbC fusions to the recombinant protein used to express proteins with disulfide bond. As well, 208-amino acid DsbA helps with forming a disulfide bond and 236-amino acid DsbC facilitates the isomerization of disulfide bond [11, 34-37]. 1.11. pHAT Vector Given that the use of six consecutive histidines as purification tags can strongly lower protein solubility, Clontech Co. introduced a vector named pHAT containing 19-amino acid tag. Six out of these 19 amino acids are histidines, but these histidines are of nonadjacent type; therefore, their overall charge is lower compared with consecutive histidines, so the solubility of proteins with pHAT fusion is higher [38, 39]. 1.12. pMAL and pGEX In some occasions, proteins with His-tag are not expressed well and they may form the inclusion body. In this case, the target gene can be cloned in vectors such as pGEX or pMAL in order to increase solubility. In this regard, the pGEX vector adds Glutathione S-transferases (GST) to the obtained protein. This tag with a weight of 25 kDa can lead to increased protein solubility. The pMAL vector also adds Maltose Binding Protein (MBP) to the obtained protein. The weight of this tag is 44 kDa. New England Biolabs Co. provides both of these vectors. The high weight of tags can be a disadvantage for these vectors. Therefore, this tag can affect the structure or function of the protein when it is not removed. In addition, a protease cleavage site has been considered in the vectors to remove the fused tag from the protein so that no additional amino acids remain in the protein. Thrombin, SUMO protease, Factor Xa, and Enterokinase are documented as the most commonly proteases used to remove a protein tag [40-44]. 1.13. pSUMO Vector SUMO tag is added to the end of proteins that have their genes cloned in pSUMO vector. Accordingly, this tag can act as a chaperonin and facilitate folding; it can also increase the solubility of proteins. LifeSensors Incorporation (Inc.) similarly constructs a variety of pSUMO vectors that can act as a shuttle vector (E.coli/yeast and E.coli/mammalian cell). The markers of these vectors are ampicillin and kanamycin. The advantage of these vectors is that the obtained protein will not have any additional amino acids after using SUMO protease [45, 46].

Hayat et al.

1.14. Medium Given that the increased expression of chaperones helps with correcting the folding of proteins, some chemicals can be used to induce the chaperones in the host. The expressed proteins can also reach to more stable status via some small chemicals or ligands. For instance; the use of ethanol, benzyl alcohol, osmolytes and/or thermal shock and changes in the buffering power can be some of the factors increasing the expression of the proteins. In addition, these factors can boost the expression of proteins in the soluble state and also elevate the stability of proteins during the purification process [4, 5, 19, 47, 48]. 2. METHODS 2.1. Length of Induction Induction is better to be performed when the cells are at the maximum concentration. Therefore, cell density is one of the factors affecting protein expression. As a result, the best time for induction is the early mid-log phase though some studies have indicated that the given induction can be also carried out in mid-log phase or stationary one. Therefore the best optical density (OD600) for induction is between 0.6-0.8 [4, 5, 19, 47-49]. 2.2. Temperature and Duration of Induction The duration and temperature of induction are two of the most important factors affecting expression and/or solubility. In this respect, decreasing the post-induction temperature can reduce the protein synthesis rate and consequently prevent the formation of inclusion bodies. This approach has been documented to be effective for some of the complicated proteins. Thus, the most commonly used temperatures for inducing poorly soluble proteins are 18°, 24° and 30°C. Although cell growth can be increased at high temperatures, it can be harmful for protein expression since the higher growth rate can cause a higher possibility of plasmid loss. This can be significant if recombinant proteins are overexpressed in continuous cultures. Generally, aggregation reaction is preferred at higher temperatures because the hydrophobic interactions are firmly dependent on temperature. Furthermore; low levels of cell division, transcription and translation, as well as decreased protein aggregation are caused by slow cell processes at the lower temperatures. In this regard, lower expression temperature reduces the degradation of proteolytically sensitive proteins because most proteases at lower temperatures are less active. Therefore, the solubility of proteins can be increased by lowering the temperature (with reducing the IPTG concentration) and increasing induction duration. Nevertheless, the optimum combination of induction duration and post-induction temperature are still obtained by trial and error [4, 5, 19, 47-49]. 2.3. Inducer Concentration Low concentrations of inducer can lead to insufficient induction of the protein expression and thereby lower efficiency. Moreover; in addition to having high costs, the high concentration of inducer can kill cells and prevent protein expression in them due to its toxic effects on cells. Therefore, regulation of inducer concentration is of utmost importance and it is better to be a little higher than critical one. According to several studies, it has been demonstrated that samples with IPTG concentrations between 0 and 1 mM cannot have toxic effects on the growth of E.coli. In addition, the use of lower concentration of inducer (e.g., 0.05 mM for IPTG) helps with expressing the protein in a soluble form, and the use of higher concentration of the inducer can reduce protein solubility. Finally, it is important to note that all the three factors of vector, host, and protein need to be considered in order to determine the appropriate concentration of inducer [4, 5, 19, 47-49].


2.4. Troubleshooting There are lots of factors disrupting the expression of host proteins. The sequence at translation initiation region (TIR), the codon usage, and the proper formation of disulfide bonds are of the pivotal factors affecting the expression of protein. The expression of the gene can be also strongly affected due to uncommon codons for E.coli. Besides, there is a possibility that target genes have codons at low abundance in this host because of the heterologous feature of the target protein which can result in premature translation termination, growth arrest, and decreased protein production yield. Additionally, there are two methods to address this issue including the use of de novo gene synthesis to substitute the rare codons in the gene sequence (a software was designed to assess the codon context or codon pair usage in addition to codon frequency), and secondly the expression of targeted gene in E.coli supplemented by tRNAs at a low level, as mentioned earlier. Moreover, it has been indicated that the protein expression levels can be strongly affected by the sequence at the 5′ of the gene because the translation can be prevented by the formation of secondary structures in the mRNA using the ribosome complex. Accordingly, protein expression can be greatly influenced by these sequences in a direct manner after the start of codon up to the position +25. Meanwhile, the eukaryotic cells have much longer mRNA halflife compared with bacteria. The mRNA stability has demonstrated to be increased by the mutation in the gene for RNaseE coding. Invitrogen Co. has presented a strain with the commercial name of BL21 Star derived from BL21 which contains respective mutation. It should be noted that a covalent disulfide bond occurs between two sulfur atoms of two cysteine residues. It is a major property to be considered in target gene expression because of its pivotal role in stability, folding, and/or target protein functioning. The formation of disulfide bonds can be seen in environments such as the bacterial periplasm or eukaryotic endoplasmic reticulum which are oxidizing environments. Thus, the functioning of DsbC system in which disulfide formation is catalyzed by DsbA is necessary in the formation of disulfide bonds in the periplasmic of E.coli, whereas isomerization of wrongly formed disulfide bonds is catalyzed by the DsbC. Nevertheless, the saturated translocation machinery can be considered as a common disadvantage of periplasmic expression which may reduce the ultimate efficiency of the target protein with its possible toxic effect on the host cell. These negative effects can be reduced as much as possible by avoiding the saturation of the translocation machinery through utilizing a strain with accurately controlled expression intensity such as Lemo21 (DE3). Accordingly, engineered E.coli strains with more oxidizing cytoplasm have been developed as substitutes for periplasmic expression to optimize the formation of disulfide bonds. These strains have mutations of thioredoxin reductase (trxB) and glutathione reductase (gor) genes which play a role in maintaining the decreased environment in the cytoplasm and a mutation in the peroxiredoxin gene ahpC that is a key factor for returning the growth in these mutants such as Origami, made in Novagen Co.. Nevertheless, lack of the isomerization of disulfide bond is considered as the major problem for utilizing these strains. Furthermore, it has been suggested that the elimination of reducing pathways in some cases can be less effective compared with the time when a catalyst is added to the formation of disulfide bonds. Meanwhile, the higher efficacy of strain BL21 (DE3) pLysS in the production of oxidized, folded, and soluble proteins compared with Shuffle T7 Express lysY or Origami B (DE3) pLysS cells has been demonstrated after utilizing 28 various small proteins with high levels of disulfide in order to form N-terminal fusions with DsbC. It was explained that the given process was performed ex vivo when the possibility of the occurrence of disulfide bond formation in the cytoplasm of BL21 (DE3) pLysS cells or during


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the purification and extraction phases was assessed using one of the fusions. One of the desirable cells used in the field of recombinant expression as a microbial cell factory has always been E.coli. E.coli can be considered as an appropriate host for the expression of globe-like proteins from prokaryotes and eukaryotes as well as the more stably folded ones. The use of this prokaryote as a precious means to produce recombinant proteins either in basic research or in commercial purposes offers many advantages. All of the abovementioned items in this article including vector, media condition, and host can influence the expression of proteins in E.coli. However, an expression system which perfectly works with all recombinant proteins cannot be found and it is necessary to obtain an optimum level of stability and high-level synthesis in each case using experimental changes in various factors because each protein can pose a new challenge. It can be concluded that an appropriate combination of the equipment of an extensive genetic toolbox can play a major role in successful preparation of the recombinant proteins in E.coli. The most important factors in this domain are illustrated in Table 3, which should be considered as significant items [5, 48, 50-53]. CONCLUSION Although much progress has been made in the field of heterologous protein expression in E.coli, expressing a protein with optimal solubility and appropriate structural and functional properties is still a problematic issue. There are numerous strategies to meet these objectives as reviewed in the above sections. In general; host strain, vector, and cultivation parameters are known as crucial parameters determining the success of recombinant protein expression in E.coli. In this regard, successful protein expression in E.coli initially necessitates a wide knowledge about physicochemical properties of the recombinant protein. In the second step, a rational selection must be made among common strains of E.coli and vectors. Finally; all factors related to media including time, temperature, and inducer need to be optimized. CONSENT FOR PUBLICATION Not applicable. CONFLICT OF INTEREST The authors declare no conflict of interest, financial or otherwise. ACKNOWLEDGEMENTS Declared none. REFERENCES [1] [2] [3]

[4] [5] [6]

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Casali N. Escherichia coli host strains. E. coli Plasmid Vectors: Methods and Applications, 2003: 27-48. Baneyx F. Recombinant protein expression in Escherichia coli. Current opinion in biotechnology, 1999; 10: 411-421. Blattner FR, Plunkett G, Bloch CA, Perna NT, Burland V, Riley M, Collado-Vides J, Glasner JD, Rode CK, Mayhew GF. The complete genome sequence of Escherichia coli K-12. science, 1997; 277: 1453-1462. Rosano GL, Ceccarelli EA. Recombinant protein expression in Escherichia coli: advances and challenges. Recombinant protein expression in microbial systems, 2014; 7. Sørensen HP, Mortensen KK. Advanced genetic strategies for recombinant protein expression in Escherichia coli. Journal of biotechnology, 2005; 115: 113-128. Chart H, Smith H, La Ragione R, Woodward MJ. An investigation into the pathogenic properties of Escherichia coli strains BLR, BL21, DH5α and EQ1. Journal of applied microbiology, 2000; 89: 1048-1058. Grodberg J, Dunn JJ. ompT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification. Journal of bacteriology, 1988; 170: 1245-1253.

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