Advanced
Production of Biopharmaceuticals in E. coli: Current Scenario and Future Perspectives
Production of Biopharmaceuticals in E. coli: Current Scenario and Future Perspectives
Journal of Microbiology and Biotechnology. 2015. Jul, 25(7): 953-962
Copyright © 2015, The Korean Society For Microbiology And Biotechnology
  • Received : December 30, 2014
  • Accepted : February 13, 2015
  • Published : July 28, 2015
Download
PDF
e-PUB
PubReader
PPT
Export by style
Share
Article
Author
Metrics
Cited by
TagCloud
About the Authors
Mohammed N. Baeshen
Department of Biological Sciences, Faculty of Science, King Abdulaziz University, Jeddah, 21589, Saudi Arabia
Ahmed M. Al-Hejin
Department of Biological Sciences, Faculty of Science, King Abdulaziz University, Jeddah, 21589, Saudi Arabia
Roop S. Bora
Department of Biotechnology, Eternal University, Baru Sahib-173 101, Himachal Pradesh, India
Mohamed M. M. Ahmed
Nucleic Acids Research Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), City for Scientific Research and Technology Applications, Alexandria 21934, Egypt
mmmahmed6@yahoo.ca
Hassan A. I. Ramadan
Cell Biology Department, Genetic Engineering and Biotechnology Division, National Research Centre, Dokki-Cairo 12311, Egypt
Kulvinder S. Saini
Department of Biotechnology, Eternal University, Baru Sahib-173 101, Himachal Pradesh, India
Nabih A. Baeshen
Department of Biological Sciences, Faculty of Science, King Abdulaziz University, Jeddah, 21589, Saudi Arabia
Elrashdy M. Redwan
Protein Research Department, Genetic Engineering and Biotechnology Research Institute, City for Scientific Research and Applied Technology, New Borg AL-Arab, Alexandria 21934, Egypt

Abstract
Escherichia coli is the most preferred microorganism to express heterologous proteins for therapeutic use, as around 30% of the approved therapeutic proteins are currently being produced using it as a host. Owing to its rapid growth, high yield of the product, costeffectiveness, and easy scale-up process, E. coli is an expression host of choice in the biotechnology industry for large-scale production of proteins, particularly non-glycosylated proteins, for therapeutic use. The availability of various E. coli expression vectors and strains, relatively easy protein folding mechanisms, and bioprocess technologies, makes it very attractive for industrial applications. However, the codon usage in E. coli and the absence of post-translational modifications, such as glycosylation, phosphorylation, and proteolytic processing, limit its use for the production of slightly complex recombinant biopharmaceuticals. Several new technological advancements in the E. coli expression system to meet the biotechnology industry requirements have been made, such as novel engineered strains, genetically modifying E. coli to possess capability to glycosylate heterologous proteins and express complex proteins, including full-length glycosylated antibodies. This review summarizes the recent advancements that may further expand the use of the E. coli expression system to produce more complex and also glycosylated proteins for therapeutic use in the future.
Keywords
Introduction
The drug discovery and development process entails the expression of large numbers of recombinant proteins in copious amounts and properly folded as 3D structures. These therapeutic recombinant proteins are generally used for the treatment of various diseases, or are used as a target protein for screening of new and novel drugs. E. coli is one of the most desirable host for the expression of several recombinant proteins owing to its rapid growth rate, easier genetic manipulations, and high level of recombinant protein synthesis rates [63 , 65] . In a few instances, very high levels of expression are achieved; that is, up to 30% of total cellular protein. Escherichia coli was the first expression host that was used for manufacturing a biopharmaceutical, which resulted in the regulatory approval of human insulin in 1982 for the treatment of diabetes. The approval of bovine growth hormone (bGH) in 1994 set a new standard for manufacturing heterologous proteins from E. coli for therapeutic use ( Table 1 ). It is even more impressive that both insulin and bovine growth hormone require oxidative protein folding, and, in addition, insulin is a heterodimer. Success achieved with recombinant insulin and growth hormone highlights the versatility and cost-effectiveness of E. coli -based production.
List of biopharmaceuticals produced inE. coli.
PPT Slide
Lager Image
All data obtained from corporate websites and http://www.fda.gov/. rh, recombinant human; G-CSF, Granulocyte-colony stimulating factor; EU, European union; US, United States of America.
However, in E. coli , there is a probability of translational errors due to the presence of a large number of rare codons in the heterologous gene(s). In the case of therapeutic proteins, these errors, even at low levels, can cause adverse immunogenic responses in humans. These cellular errors during protein translation may impact the tertiary structure and thus affect the biological activity of the recombinant protein. In this review, we will be focusing on the “desired modulation” of key biochemical parameters required to obtain properly folded and overexpressed biologically active therapeutic proteins in E. coli .
Codon Usage inE. coli
In E. coli , codon usage is manifested by the level of cognate aminoacylated tRNAs in the cytosol. Major codons are generally present in genes that are expressed at a high level; on the other hand, rare codons are commonly encountered in genes that are expressed at low levels. Codons that are rare in E. coli are found to be abundant in eukaryotic genes [31] . Expression of heterologous genes harboring rare codons can result in translational errors, due to ribosomal stalling at positions where amino acids coupled to rare codon tRNAs have to be incorporated [43] . In addition, translational errors due to the presence of rare codons in heterologous genes might include amino acid substitutions, frame-shift mutations, or premature termination of translation [31 , 69] . Kane et al . [30] had reported inframe, two amino acids “hops” at a rare AGA codon. It had also been reported that protein quality is affected to a great extent by codon bias, due to the incorporation of lysine for arginine at the AGA codon [4 , 67] . Hence, expression of recombinant proteins, even at very high levels, is of no use if the quality of the protein is compromised as a result of translational errors. The most problematic rare codons in E. coli are AGA, AGG, CGG, CGA, CGG (arginine), AUA (isoleucine), GGA (glycine), CUA (leucine), CCC (proline), and AAG (lysine) [85] . It has been documented that rare arginine codons, AGG and AGA, occur at frequencies of 0.14% and 0.21%, respectively, in E. coli [31] .
Several strategies are employed to circumvent the issue of codon bias in E. coli . One approach is to synthesize the whole gene based on codon usage, which is currently a preferred method to improve the expression of heterologous proteins in E. coli [59 , 61] . However, a major drawback is the high cost associated with total gene synthesis. Another approach involves the site-directed mutagenesis of the heterologous gene sequence to generate codons, which reflects the tRNA pool of E. coli . However, this process may become very tedious and expensive, whenever several nucleotides need to be modified. Another strategy requires the co-transformation of E. coli strains with plasmid harboring a gene encoding the tRNA cognate to the rare codons [12] . By enhancing the copy number of the limiting tRNAs, E. coli can be manipulated to match the codon usage frequency in heterologous genes. Several commercial plasmids, such as pRARE, are now available for rare tRNA coexpression in E. coli . Moreover, these plasmids contain the p15A replication origin, which enables their maintenance in the presence of the ColE1 replication origin in the E. coli expression vectors. There are also several commercial E. coli strains available, as listed in Table 2 , that harbor plasmids containing gene sequences encoding tRNA for rare codons, such as BL21(DE3) CodonPlus-RIL, BL21(DE3) CodonPlus-RP (Stratagene, USA), and Rosetta (DE3). Several studies have employed these excellent strategies to enhance the expression of heterologous proteins in E. coli . [13 , 33 , 45 , 46 , 71] . This approach has been successfully used to enhance the industrial production of human recombinant interferon. Co-transformation of E. coli BL21 (DE3) with the plasmid harboring human interferon-α2a gene and the argU gene that encodes for rare tRNAs for Arg (AGG/AGA) resulted in much higher levels of expression of IFN-α2a, and the recombinant protein constituted about 25% of the total proteins [26] . In another study, it was observed that production of human interferon alpha 2b protein was increased 9.5-11.5-fold by replacing the rare arginine codons with more frequently used codons [78] . The E. coli Rosetta (DE3) strain containing plasmid pRARE was used to express different human proteins, and it was shown that the yields of the recombinant protein were increased dramatically for about 35 of the 68 proteins tested [17 , 75] .
Commercially availableE. colistrains to improve protein solubility.
PPT Slide
Lager Image
Source: http://wolfson.huji.ac.il/expression/bac-strains-prot-exp.html http://www.emdmillipore.com/life-science-research/novagen http://www.invitrogen.com/1/3/stratagene-products https://www.neb.com https://www.lucigen.com https://www.genlantis.com
Protein Translation
Efficient translation initiation in E. coli requires a ribosomal binding site that includes the Shine-Dalgarno (SD) sequence and a translation initiation codon [69] . The Shine-Dalgarno sequence is generally located 7-9 nucleotides upstream from the initiation codon AUG [62] . It has been observed that translation initiation is more efficient from mRNAs containing the consensus SD sequence AAGGAGG. The secondary structure of ribosomal binding site is very critical for translation initiation, and the translation efficiency is further increased by the presence of a large number of thymine and adenine [35] . The efficiency of translation initiation is also affected by the nucleotide that follows the initiation codon, and it has been observed that adenine is very common in highly expressed genes [74] . Taken together, translation initiation is affected by various factors, including a consensus Shine-Dalgarno sequence, nucleotides upstream of the initiation codon, and the secondary structure of the ribosomal binding site [73] . It had also been reported that mRNA secondary structures that prevent ribosome binding might affect the protein expression to a great extent [11 , 14] . It was observed that a single base change compromised the stability of the secondary structure near the SD region and resulted in a 500-fold change in the expression levels of the coat protein of RNA bacteriophage MS2. Park et al . [55 , 68] have developed a very efficient and simple method for designing 5’-untranslated region (5’UTR) variants for tunable expression in E. coli . Since 5’UTR containing the SD sequence and the AU-rich sequence play a significant role in protein translation, the expression levels of recombinant proteins can be manipulated by incorporating simple variations in the 5’UTR. It has been found that secondary structures in mRNA could be disrupted by RNA helicases such as the DEAD protein of E. coli . It was shown that expression of the DEAD protein enhanced the expression of β-galactosidase by 30-fold from the T7 promoter, but there was no significant increase in the expression levels from the lac promoter [22] . However, in the absence of DEAD protein, β-galactosidase synthesis from a T7 promoter was found to be 10-fold less as compared with the expression from the lac promoter, even though the transcription rate was 10-fold higher. It was proposed that the DEAD-box protein plays an important role in stabilizing the mRNA [6 , 23 , 39] . The DEAD-box protein thus can be exploited to additionally improve the expression of genes with suspected problematic mRNA secondary structures.
In the bacterial genome, UAA is the most frequently used termination codon, followed by UGA and UAG. During translation, an error in reading the termination codon leads to extended protein synthesis until another termination codon is encountered in the mRNA. This read-through results in the synthesis of a larger peptide, with several additional C-terminal amino acids. It had been shown that replacing UGA with the UAA terminator in human IFN-α2b resulted in a 2-fold increase in protein expression level [66] . It had been shown that transcription terminators stabilize the mRNA by creating a stem loop structure at the 3’ end of the mRNA [49] . In E. coli , stop codon UAA is used more commonly for translation termination. The efficiency of translation termination can be improved by adding consecutive stop codons or by using a prolonged UAAU stop codon [57] . Translation errors during protein synthesis can cause frame-shift mutations, premature truncation, lower expression, and misincorporation of amino acid, and thus adversely affect the quality of recombinant protein production in E. coli [3 , 56 , 67] . High expression levels and correct apparent molecular mass does not always ensure the translation integrity of the recombinant protein [4 , 18 , 52] .
Molecular Chaperones to Optimize Protein Folding
One major issue in the production of biopharmaceuticals using E. coli host is the accumulation of heterologous proteins, mainly as insoluble aggregates in the form of inclusion bodies. Extraction of recombinant protein from these inclusion bodies is a tedious and cumbersome process that requires denaturation and several renaturing steps to obtain a soluble and properly folded recombinant protein. One strategy to improve protein solubility in E. coli is the use of molecular chaperones. Inclusion bodies in E. coli comprise misfolded aggregates of heterologous proteins. During protein synthesis, molecular chaperones interact with nascent polypeptide chains to prevent aggregation during the folding process. Some chaperones are shown to prevent protein aggregation, while other chaperones promote refolding and solubilization of misfolded proteins [5] . The most widely used and important cytoplasmic chaperones in E. coli are DnaK, DnaJ, GrpE, GroEL, GroES, and Trigger factor ( Table 3 ). These chaperones have been used either singly or in combination to improve the protein solubility in E. coli [1 , 9 , 10 , 38 , 64] . The most efficient chaperone combinations that are widely used to improve protein refolding are GroEL-GroES and DnaK-DnaJ-GrpE [27 , 50 , 84 , 80] . In addition, it was observed that DnaKDnaJ-GrpE assists in the release of unfolded proteins and GroEL-GroES chaperones prevent the degradation of peptides [72] . Trigger factor has been shown to interact with GroEL and improves GroEL-substrate binding to facilitate protein folding [19] . Other chaperones such as ClpB (Hsp100), in association with DnaK, solubilizes protein and prevents aggregation [2 , 47] . Other heat shock proteins, such as IpbA and IpbB, prevent aggregation of heat denatured proteins [20] . For improving the production of recombinant heterologous proteins in E. coli , different combinations of molecular chaperones should be analyzed to identify the most efficient one. It has been shown that coexpression of Skp and FkpA chaperones increased the solubility of antibody fragments in E. coli [53] . It was revealed that coexpression of GroEL–GroES resulted in production of about 65% of anti-B-type natriuretic peptide single chain antibody (scFv) in soluble form, which was 2.4-fold higher than in the absence of chaperones [40] .
Application of molecular chaperones to improve protein solubility inE. coli.
PPT Slide
Lager Image
ALDH3A1, aldehyde dehydrogenase 3 family, member A1; xynB, xylanase B; IL-6, Interleukin-6; Fab, fragment antigen-binding; HSP, heat shock protein.
Another strategy involves the lowering of the growth temperature to enhance the solubility of recombinant proteins [32] . Growth at a lower temperature decreases the rate of protein synthesis and thus prevents the accumulation of folding intermediates in the cytosol. Moreover, it also reduces the aggregation of protein by preventing inter- and intramolecular hydrophobic interactions, thus minimizing the formation of inclusion bodies [15] . This approach has been very efficient in enhancing the solubility of various therapeutic proteins such as interferon-α-2, human growth hormones, and Fab fragments [24 , 79] . In addition, to facilitate the production of soluble recombinant proteins in E. coli , it is crucial to optimize several other parameters, such as effect of medium composition, choice of expression vectors, choice of promoters, choice of expression hosts, application of various fusion tags, rate of protein synthesis, inducer concentration, and duration of induction [16 , 36 , 54] .
It has been suggested that the secretion of heterologous proteins into the periplasm of E. coli may provide an opportunity to produce complex therapeutic proteins. The Dsb protein family in the periplasm assists in disulfide bond formation, and the oxidizing environment in the periplasm facilitates the proper folding of heterologous proteins. Moreover, the periplasm contains very few host protein and low proteolytic activity, which facilitate the downstream processing to recover high yields of therapeutic proteins [41] . Various therapeutic proteins have been successfully manufactured by periplasmic secretion, such as commercialized Fab fragments ( i.e. , Leucentis and Cimza) and some full-length aglycosylated antibodies and scFvs [29 , 48] . Various secretion signals have been used to target heterologous proteins to the periplasm of E. coli , such as Omp A, LamB, PhoA, STII, PelB, and endoxylanase [7] . Yim et al . [86] exploited the endoxylanase signal peptide to obtain very high-levels of expression of granulocyte colonystimulating factor (G-CSF) at 4.2 g/l in the periplasm of E. coli . Another study reported high-level periplasmic expression of insulin-like growth factor I (IGF-1), with a yield of 4.3 g/l, by using the LamB signal peptide [28] . It must be noted that besides signal peptides, the efficiency of periplasmic secretion also depends on the E. coli strain, promoter strength, and growth temperature. In some circumstances, coexpression of periplasmic chaperones, including disulfide bond oxidase (DsbA), isomerase (DsbC), and peptidylprolyl isomerase, could enhance the production of heterologous proteins in E. coli [34] . Reilly and Yansura [58] reported that overexpression of DsbA and DsbC improved the efficiency of the assembly of the light chain and the heavy chain of a full-length antibody in the periplasm, and increased the production from 0.1 to 1.05 g/l [58] . Another study reported that the yield of anti-CD20 scFv was increased, along with improved antigen binding affinity, when it was coexpressed with the periplasmic chaperone Skp [42] . Lee et al . [37] reported the development of an efficient expression system for full-length IgG in E. coli . Their study demonstrated that modification of the 5’UTR sequence and coexpression of DsbC foldase resulted in very high expression levels of heavy and light chains, and assembly of IgG was also dramatically improved in the periplasm. Under highly optimized condition, fully assembled and functionally active IgG was produced, as high as 362 mg/ml [37] . These studies clearly suggested that through proper engineering of the E. coli host, it would be possible to produce therapeutic monoclonal antibodies in E. coli in a very cost-effective and timely manner in the near future.
Post-Translational Modifications
E. coli is a favorite microorganism of biotechnologists for the large-scale production of therapeutic proteins [60] . However, the absence of post-translational modification processes in E. coli limits its use for the production of recombinant biopharmaceuticals. Various post-translational modifications, including glycosylation and phosphorylation, which are critical for functional activity, do not take place in E. coli due to its lack of such cellular machinery [25 , 82] . N-Linked glycosylation of proteins is one of the most important post-translational modifications in eukaryotes. Wacker et al . [81] identified a novel N-linked glycosylation pathway in the bacterium Campylobacter jejuni and also showed the successful transfer of a functionally active Nglycosylation pathway into E. coli [81] . Campylobacter jejuni harbors pgl gene clusters, which are involved in the synthesis of various glycoproteins. By successfully transferring the pgl pathway into E. coli , various glycosylated proteins were produced in E. coli . Although the bacterial N-glycan structure is not similar to that seen in eukaryotes, the molecular engineering of the glycosylation pathway of C. jejuni into E. coli has paved the way for expressing glycosylated proteins in E. coli [77 , 51] . Valderrama et al . [77] successfully engineered the eukaryotic glycosylation pathway in E. coli . Four eukaryotic glycosyltransferases, which include yeast uridine diphosphate-N-acetylglucosamine transferases Alg13 and Alg14, mannosyl-transferases Alg1 and Alg2, and bacterial oligosaccharyltransferase PglB from C. jejuni , were coexpressed in E. coli for synthesizing glycans, which were then successfully transferred to asparagine residues in the target eukaryotic protein [77] . This approach can also be utilized to develop glycoconjugate vaccines against several bacterial pathogens, which could be a more cost-effective and convenient alternative method to presently employed chemical-based methods of vaccine production. Currently, a glycoconjugate vaccine against Shigella dysenteriae O1, developed using this technology, has successfully cleared the phase I clinical trials. Initial efficacy and safety studies demonstrated that the glycoconjugate vaccine was safe and also elicited a strong immune response. This novel approach for glycoconjugate vaccine production using the engineered N-linked glycosylation system of Campylobacter jejuni can be exploited to produce vaccines against both gram-positive and gram-negative pathogens [8 , 18 , 21 , 44 , 83] .
Future Perspectives
Although there are several different expression systems such as yeast, mammalian cell lines, transgenic animals, and plants that are currently being used for the production of recombinant proteins, new technological advancements are continuously being made to improve the E. coli expression system. Production of heterologous proteins in E. coli is always preferred because of the ease of genetic manipulations, well-characterized genome, accessibility of versatile plasmid vector, availability of different kinds of host strains, cost-effectiveness, and very high expression levels as compared with other expression systems. However, there are certain limitations that might hinder the efficient use of the E. coli system to overexpress heterologous genes, such as biased codon usage, protein solubility, mRNA stability, and secondary structure. The translational errors due to the presence of rare codons in heterologous proteins may result in amino acid substitutions or frame-shift mutations, which ultimately leads to undesired products. Hence, the codon usage of the recombinant protein plays a critical role in defining its expression levels. Expression of therapeutic proteins in E. coli can be enhanced by replacing rare codons in the genes with more favorable major codons. Similarly, the coexpression of genes coding for tRNAs for rare codons could increase the expression levels of therapeutic proteins in E. coli . In addition, periplasmic secretion of heterologous proteins offers several advantages, including proper folding, solubility, ease in purification, and higher yield of proteins. Antibody fragments that have been approved for therapeutic use were produced in the periplasm of E. coli , suggesting that this approach is commercially viable. Various recent studies have shown that E. coli strains can be modified specifically for each therapeutic protein to achieve high product yields as well as high-quality products.
Acknowledgements
This work was supported by the NSTIP strategic technologies program in the Kingdom of Saudi Arabia (Project No. 10-BIO1257-03). The authors also acknowledge financial assistance from the Science & Technology Unit, Deanship of Scientific Research, and Deanship of Graduate Studies, King Abdulaziz University, Jeddah, KSA.
References
Arya R , Sabir JSM , Bora RS , Saini KS 2015 Optimization of culture parameters and novel strategies to improve protein solubility. In Insoluble Proteins: Methods and Protocols. Methods in Molecular Biology series, edited by Dr.Elena Garcia Fruitos, Springer Science and Business Media New York, USA Vol. 1258, 3 45 - 63
Betiku E 2006 Molecular chaperones involved in heterologous protein folding inEscherichia coli. Biotechnol. Mol. Biol. 1 66 - 75
Brinkman U , Mattes RE , Buckel P 1989 High-level expression of recombinant genes inEscherichia coliis dependent on the availability of the dnaY gene product. Gene 85 109 - 114    DOI : 10.1016/0378-1119(89)90470-8
Calderone TL , Stevens RD , Oas TG 1996 High-level misincorporation of lysine for arginine at AGA codons in a fusion protein expressed inEscherichia coli. J. Mol. Biol. 262 407 - 412    DOI : 10.1006/jmbi.1996.0524
Carrio MM , Villaverde A 2003 Role of molecular chaperones in inclusion body formation. FEBS Lett. 537 215 - 221    DOI : 10.1016/S0014-5793(03)00126-1
Chen R 2012 Bacterial expression systems for recombinant protein production:E. coliand beyond. Biotechnol. Adv. 30 1102 - 1107    DOI : 10.1016/j.biotechadv.2011.09.013
Choi JH , Lee SY 2004 Secretory and extracellular production of recombinant proteins usingEscherichia coli. Appl. Microbiol. Biotechnol. 64 625 - 635    DOI : 10.1007/s00253-004-1559-9
Cuccui J , Wren B 2015 Hijacking bacterial glycosylation for the production of glycoconjugates, from vaccines to humanised glycoproteins. J. Pharm. Pharmacol. 67 338 - 350    DOI : 10.1111/jphp.12321
Cui SS , Lin XZ , Shen JH 2011 Effects of co-expression of molecular chaperones on heterologous soluble expression of the cold-active lipase Lip-948. Protein Expr. Purif. 77 166 - 172    DOI : 10.1016/j.pep.2011.01.009
de Marco A 2007 Protocol for preparing proteins with improved solubility by co-expressing with molecular chaperones inEscherichia coli. Nat. Protoc. 2 2632 - 2639    DOI : 10.1038/nprot.2007.400
De Smit MH , van Duin J 1990 Secondary structure of the ribosome binding site determines translational efficiency: a quantitative analysis. Proc. Natl. Acad. Sci. USA 87 7668 - 7672    DOI : 10.1073/pnas.87.19.7668
Dieci G , Bottarelli L , Ballabeni A , Ottonello S 2000 tRNA assisted overproduction of eukaryotic ribosomal proteins. Protein Expr. Purif. 18 346 - 354    DOI : 10.1006/prep.2000.1203
El-Baky NA , Redwan EM 2015 Therapeutic alpha-interferons protein: structure, production, and biosimilar. Prep. Biochem. Biotechnol. 45 109 - 127    DOI : 10.1080/10826068.2014.907175
Etchegaray JP , Inouye M 1999 Translational enhancement by an element example of molecular misreading in Alzheimer’s disease. Trends Neurosci. 21 331 - 335
Fahnert B , Lilie H , Neubauer P 2004 Inclusion bodies: formation and utilization. Adv. Biochem. Eng. Biotechnol. 89 93 - 142
Feng Y , Xu Q , Yang T , Sun E , Li J , Shi D , Wu D 2014 A novel self-cleavage system for production of soluble recombinant protein inEscherichia coli. Protein Expr. Purif. 99 64 - 69    DOI : 10.1016/j.pep.2014.04.001
Ferrer-Miralles N , Villaverde A 2013 Bacterial cell factories for recombinant protein production; expanding the catalogue. Microb. Cell Fact. 12 113 -    DOI : 10.1186/1475-2859-12-113
Fisher AC , Haitjema CH , Guarino C , Celik E , Endicott CE , Reading CA 2011 Production of secretory and extracellular N-linked glycoproteins inEscherichia coli. Appl. Environ. Microbiol. 77 871 - 881    DOI : 10.1128/AEM.01901-10
Folwarczna J , Moravec T , Plchova H , Hoffmeisterova H , Cerovska N 2012 Efficient expression of human papillomavirus 16 E7 oncoprotein fused to C-terminus of tobacco mosaic virus (TMV) coat protein using molecular chaperones inEscherichia coli. Protein Expr. Purif. 85 152 - 157    DOI : 10.1016/j.pep.2012.07.008
Guzzo J 2012 Biotechnical applications of small heat shock proteins from bacteria. Int. J. Biochem. Cell Biol. 44 1698 - 1705    DOI : 10.1016/j.biocel.2012.06.007
Ihssen J , Kowarik M , Dilettoso S , Tanner C , Wacker M , Thöny-Meyer L 2010 Production of glycoprotein vaccines inEscherichia coli. Microb. Cell Fact. 11 (9) 61 -    DOI : 10.1186/1475-2859-9-61
Iost I , Dreyfus M 1994 mRNAs can be stabilized by DEAD-box proteins. Nature 372 193 - 196    DOI : 10.1038/372193a0
Iost I , Bizebard T , Dreyfus M 2013 Functions of DEAD-box proteins in bacteria: current knowledge and pending questions. Biochim. Biophys. Acta 1829 866 - 877    DOI : 10.1016/j.bbagrm.2013.01.012
Jensen EB , Carlsen S 1990 Production of recombinant human growth hormone inEscherichia coli: expression of different precursors and physiological effects of glucose, acetate and salts. Biotechnol. Bioeng. 36 1 - 11    DOI : 10.1002/bit.260360102
Jenkins N 2007 Modifications of therapeutic proteins: challenges and prospects. Cytotechnology 53 121 - 125    DOI : 10.1007/s10616-007-9075-2
Jeong W , Shin HC 1998 Supply of the ArgU gene product allows high-level expression of recombinant human interferonalpha-2a inEscherichia coli. Biotechnol. Lett. 20 19 - 22    DOI : 10.1023/A:1005366727366
Jhamb K , Sahoo DK 2012 Production of soluble recombinant proteins inEscherichia coli: effects of process conditions and chaperone co-expression on cell growth and production of xylanase. Bioresour. Technol. 123 135 - 143    DOI : 10.1016/j.biortech.2012.07.011
Joly JC , Leung WS , Swartz JR 1998 Overexpression of Escherichia coli oxidoreductases increases recombinant insulinlike growth factor-I accumulation. Proc. Natl. Acad. Sci. USA 95 2773 - 2777    DOI : 10.1073/pnas.95.6.2773
Jung ST , Kang TH , Kelton W , Georgiou G 2011 Bypassing glycosylation: engineering aglycosylated full-length IgG antibodies for human therapy. Curr. Opin. Biotechnol. 22 858 - 867    DOI : 10.1016/j.copbio.2011.03.002
Kane JF , Violand BN , Curran DF , Staten NR , Duffin KL , Bogosian G 1992 Novel in-frame two codon translational hop during synthesis of bovine placental lactogen in a recombinant strain ofEscherichia coli. Nucleic Acids Res. 20 6707 - 6712    DOI : 10.1093/nar/20.24.6707
Kane JF 1995 Effects of rare codon clusters on high-level expression of heterologous proteins inEscherichia coli. Curr. Opin. Biotechnol. 6 494 - 500    DOI : 10.1016/0958-1669(95)80082-4
Khattar SK , Kundu PK , Gulati P , Singh V , Bughani U , Bajpaim M , Saini KS 2007 Optimization and enhanced soluble production of biologically active recombinant human p38 mitogen-activated-protein kinase (MAPK) inEscherichia coli. Protein Peptide Lett. 14 756 - 760    DOI : 10.2174/092986607781483660
Kim R , Sandler SJ , Goldman S , Yokota H , Clark AJ , Kim SH 1998 Overexpression of archaeal proteins inEscherichia coli. Biotechnol. Lett. 20 207 - 210    DOI : 10.1023/A:1005305330517
Kolaj O , Spada S , Robin S , Wall JG 2009 Use of folding modulators to improve heterologous protein production inEscherichia coli. Microb. Cell Fact. 8 9 -    DOI : 10.1186/1475-2859-8-9
Laursen BS , Sorensen HP , Mortensen KK , Sperling-Petersen HU 2005 Initiation of protein synthesis in bacteria. Microbiol. Mol. Biol. Rev. 69 101 - 123    DOI : 10.1128/MMBR.69.1.101-123.2005
Lebendiker M , Danieli T 2014 Production of prone-toaggregate proteins. FEBS Lett. 588 236 - 246    DOI : 10.1016/j.febslet.2013.10.044
Lee YJ , Lee DH , Jeong KJ 2014 Enhanced production of human full-length immunoglobulin G1 in the periplasm ofEscherichia coli. Appl. Microbiol. Biotechnol. 98 1237 - 1246    DOI : 10.1007/s00253-013-5390-z
Levy R , Weiss R , Chen G , Iverson BL , Georgiou G 2001 Production of correctly folded Fab antibody fragment in the cytoplasm ofEscherichia colitrxB gor mutants via the coexpression of molecular chaperones. Protein Expr. Purif. 23 338 - 347    DOI : 10.1006/prep.2001.1520
Linder P , Daugeron M-C 2000 Are DEAD-box proteins becoming respectable helicases? Nat. Struct. Biol. 7 97 - 99    DOI : 10.1038/72464
Maeng BH , Nam DH , Kim YH 2011 Coexpression of molecular chaperones to enhance functional expression of anti-BNPscFv in the cytoplasm ofEscherichia colifor the detection of B-type natriuretic peptide. World J. Microbiol. Biotechnol. 27 1391 - 1398    DOI : 10.1007/s11274-010-0590-5
Makrides SC 1996 Strategies for achieving high-level expression of genes inEscherichia coli. Microbiol. Rev. 60 512 - 538
Mavrangelos C , Thiel M , Adamson PJ , Millard DJ , Nobbs S , Zola H , Nicholson IC 2001 Increas ed yield a nd activity of soluble single-chain antibody fragments by combining highlevel expression and the Skp periplasmic chaperonin. Protein Expr. Purif. 23 289 - 295    DOI : 10.1006/prep.2001.1506
McNulty DE , Claffee BA , Huddleston MJ , Kane JF 2003 Mistranslational errors associated with the rare arginine codon CGG inEscherichia coli. Protein Expr. Purif. 27 365 - 374    DOI : 10.1016/S1046-5928(02)00610-1
Merritt JH , Ollis AA , Fisher AC , DeLisa MP 2013 Glycansby-design: engineering bacteria for the biosynthesis of complex glycans and glycoconjugates. Biotechnol. Bioeng. 110 1550 - 1564    DOI : 10.1002/bit.24885
Mohammed Y , El-Baky NA , Redwan NA , Redwan EM 2012 Expression of human interferon-α8 synthetic gene under P(BAD) promoter. Biochemistry (Mosc.) 77 1210 - 1219    DOI : 10.1134/S0006297912100136
Mohammed Y , El-Bakym NA , Redwan EM 2012 Expression, purification, and characterization of recombinant human consensus interferon-alpha inEscherichia coliunder λP(L) promoter. Prep. Biochem. Biotechnol. 42 426 - 447    DOI : 10.1080/10826068.2011.637600
Nausch H , Huckauf J , Koslowski R , Meyer U , Broer I , Mikschofsky H 2013 Recombinant production of human interleukin 6 inEscherichia coli. PLoS One 8 e54933 -    DOI : 10.1371/journal.pone.0054933
Nelson AL , Reichert JM 2009 Development trends for therapeutic antibody fragments. Nat. Biotechnol. 27 331 - 337    DOI : 10.1038/nbt0409-331
Newbury SF , Smith NH , Robinson EC , Hiles ID , Higgins CF 1987 Stabilization of translationally active mRNA by prokaryotic REP sequences. Cell 48 297 - 310    DOI : 10.1016/0092-8674(87)90433-8
Nishihara K , Kanemori M , Kitagawa M , Yanagi H , Yura T 1998 Chaperone coexpression plasmids: differential and synergistic roles of DnaK-DnaJ-GrpE and GroEL-GroES in assisting folding of an allergen of Japanese cedar pollen, Cryj2, inEscherichia coli. Appl. Environ. Microbiol. 64 1694 - 1699
Ollis AA , Zhang S , Fisher AC , DeLisa MP 2014 Engineered oligosaccharyltransferases with greatly relaxed acceptor-site specificity. Nat. Chem. Biol. 10 816 - 822    DOI : 10.1038/nchembio.1609
Overton TW 2014 Recombinant protein production in bacterial hosts. Drug Discov. Today 19 590 - 601    DOI : 10.1016/j.drudis.2013.11.008
Ow DSW , Lim DYX , Nissom PM , Camattari A , Wong VVT 2010 Co-expression of Skp and FkpA chaperones improves cell viability and alters the global expression of stress response genes during scFvD1.3 production. Microb. Cell Fact. 9 22 -    DOI : 10.1186/1475-2859-9-22
Papaneophytou CP , Kontopidis G 2014 Statistical approaches to maximize recombinant protein expression inEscherichia coli: a general review. Protein Expr. Purif. 94 22 - 32    DOI : 10.1016/j.pep.2013.10.016
Park YS , Seo SW , Hwang S , Chu HS , Ahn JH , Kim TW 2007 Design of 5’-untranslated region variants for tunable expression inEscherichia coli. Biochem. Biophys. Res. Commun. 356 136 - 141    DOI : 10.1016/j.bbrc.2007.02.127
Parker J 1989 Errors and alternatives in reading the universal genetics code. Microbiol. Rev. 53 273 - 298
Poole ES , Brown CM , Tate WP 1995 The identity of the base following the stop codon determines the efficiency of in vivo translational termination inEscherichia coli. EMBO J. 14 151 - 158
Reilly DE , Yansura DG 2010 Production of monoclonal antibodies inE. coli, pp295-308.InShire SJ, Gombotz W, Bechtold-Peters K, Andya J. (eds).Current Trends in Monoclonal Antibodies Development and Manufacturing. Springer New York
Redwan EM 2006 Optimal gene sequence for optimal protein expression inEscherichia coli: principle requirements. Arab. J. Biotechnol. 11 493 - 510
Redwan EM 2007 Cumulative updating of approved biopharmaceuticals. Hum. Antibodies 16 137 - 158
Redwan EM , Matar SM , El-Aziz GA , Serour EA 2008 Synthesis of the human insulin gene: protein expression, scaling up and bioactivity. Prep. Biochem. Biotechnol. 38 24 - 39    DOI : 10.1080/10826060701774312
Ringquist S , Shinedling S , Barrick D , Green L , Binkley J , Stormo GD , Gold L 1992 Translation initiation inEscherichia coli: sequences within the ribosome-binding site. Mol. Microbiol. 6 1219 - 1229    DOI : 10.1111/j.1365-2958.1992.tb01561.x
Rodriguez V , Asenjo JA , Andrews BA 2014 Design and implementation of a high yield production system for recombinant expression of peptides. Microb. Cell Fact. 13 65 -    DOI : 10.1186/1475-2859-13-65
Ronez F , Arbault P , Guzzo J 2012 Co-expression of the small heat shock protein, Lo18, with β-glucosidase inEscherichia coliimproves solubilization and reveals various associations with overproduced heterologous protein, GroEL/ES. Biotechnol. Lett. 34 935 - 939    DOI : 10.1007/s10529-012-0854-2
Sahdev S , Khattar SK , Saini KS 2008 Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies. Mol. Cell. Biochem. 307 249 - 264    DOI : 10.1007/s11010-007-9603-6
Sanchez JC , Padron G , Santana H , Herrera L 1998 Elimination of an HuIFN alpha 2b readthrough species, produced inEscherichia coli, by replacing its natural translation stop signal. J. Biotechnol. 63 179 - 186    DOI : 10.1016/S0168-1656(98)00073-X
Seetharam R , Heeren RA , Wong EY , Braford SR , Klein BK , Aykent S 1988 Mistranslation in IGF-1 during overexpression of the protein inEscherichia coliusing a synthetic gene containing low frequency codons. Biochem. Biophys. Res. Commun. 155 518 - 523    DOI : 10.1016/S0006-291X(88)81117-3
Seo SW , Yang JS , Cho HS , Yang J , Kim SC , Park JM 2014 Predictive combinatorial design of mRNA translation initiation regions for systematic optimization of gene expression levels. Sci. Rep. 4 4515 -
Sørensen HP , Laursen BS , Mortensen KK , Sperling-Petersen HU 2002 Bacterial translation initiation — mechanism and regulation. Recent Res. Dev. Biophys. Biochem. 2 243 - 270
Sørensen HP , Sperling-Petersen HU , Mortensen KK 2003 A favorable solubility partner for the recombinant expression of streptavidin. Protein Expr. Purif. 32 252 - 259    DOI : 10.1016/j.pep.2003.07.001
Sørensen HP , Sperling-Petersen HU , Mortensen KK 2003 Dialysis strategies for protein refolding: preparative streptavidin production. Protein Expr. Purif. 31 149 - 154    DOI : 10.1016/S1046-5928(03)00133-5
Sorensen HP , Mortensen KK 2005 Soluble expression of recombinant proteins in the cytoplasm ofEscherichia coli. Microb. Cell Fact. 4 1 -    DOI : 10.1186/1475-2859-4-1
Sprengart ML , Porter AG 1997 Functional importance of RNA interactions in selection of translation initiation codons. Mol. Microbiol. 24 19 - 28    DOI : 10.1046/j.1365-2958.1997.3161684.x
Stenstrom C , Jin H , Major L , Tate W , Isaksson LA 2001 Codon bias at the 3’-side of the initiation codon is correlated with translation initiation efficiency inEscherichia coli. Gene 263 273 - 284    DOI : 10.1016/S0378-1119(00)00550-3
Tegel H , Tourle S , Ottosson J , Persson A 2010 Increased levels of recombinant human proteins with theEscherichia colistrain Rosetta (DE3). Protein Expr. Purif. 69 159 - 167    DOI : 10.1016/j.pep.2009.08.017
Terra VS , Mills DC , Yates LE , Abouelhadid S , Cuccui J , Wren BW 2012 Recent developments in bacterial protein glycan coupling technology and glycoconjugate vaccine design. J. Med. Microbiol. 61 919 - 926    DOI : 10.1099/jmm.0.039438-0
Valderrama-Rincon JD , Fisher AC , Merritt JH , Fan YY , Reading CA , Chhiba K 2012 An engineered eukaryotic protein glycosylation pathway inEscherichia coli. Nat. Chem. Biol. 8 434 - 436    DOI : 10.1038/nchembio.921
Valente CA , Prazeres DMF , Cabral JMS , Monteriro GA 2004 Translation feature of human alpha 2b interferon production inEscherichia coli. Appl. Environ. Microbiol. 70 5033 - 5036    DOI : 10.1128/AEM.70.8.5033-5036.2004
Vasina JA , Baneyx F 1997 Expression of aggregation prone recombinant proteins at low temperatures: a comparative study of theEscherichia coli cspAandtacpromoters systems. Protein Expr. Purif. 9 211 - 218    DOI : 10.1006/prep.1996.0678
Voulgaridou GP , Mantso T , Chlichlia K , Panayiotidis MI , Pappa A 2013 EfficientE. coliexpression strategies for production of soluble human crystallin ALDH3A1. Plos One 8 e56582 -    DOI : 10.1371/journal.pone.0056582
Wacker M , Linton D , Hitchen PG , Nita-Lazar M , Haslam SM , North SJ 2002 N-linked glycosylation in Campylobacter jejuni and its functional transfer intoE. coli. Science 298 1790 - 1793    DOI : 10.1126/science.298.5599.1790
Walsh G , Jefferis R 2006 Post-translational modifications in the context of therapeutic proteins. Nat. Biotechnol. 24 1241 - 1252    DOI : 10.1038/nbt1252
Wetter M , Kowarik M , Steffen M , Carranza P , Corradin G , Wacker M 2013 Engineering, conjugation, and immunogenicity assessment ofEscherichia coliO121 O antigen for its potential use as a typhoid vaccine component. Glycoconj. J. 30 511 - 522    DOI : 10.1007/s10719-012-9451-9
Yan X , Hu S , Guan YX , Yao SJ 2012 Coexpres sion of chaperonin GroEL/GroES markedly enhanced soluble and functional expression of recombinant human interferongamma inEscherichia coli. Appl. Microbiol. Biotechnol. 93 1065 - 1074    DOI : 10.1007/s00253-011-3599-2
Yarian C , Marszalek M , Sochacka E , Malkiewicz A , Guenther R , Miskiewicz A , Agris PF 2000 Modified nucleoside dependent Watson-Crick and wobble codon binding by tRNALysUUU species. Biochemistry 39 13390 - 13395    DOI : 10.1021/bi001302g
Yim S , Jeong K , Chang H , Lee S 2001 High-level secretory production of human granulocytes-colony stimulating factor by fed-batch culture of recombinantEscherichia coli. Bioprocess Biosyst. Eng. 24 249 - 254    DOI : 10.1007/s004490100267