Recombinant protein expression in Escherichia coli: advances and challenges.

doi:10.3389/FMICB.2014.00172

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Recombinant protein expression in Escherichia coli: advances and challenges.

Journal Article•DOI•

Recombinant protein expression in Escherichia coli: advances and challenges.

Germán L. Rosano¹, Eduardo A. Ceccarelli¹•Institutions (1)

National University of Rosario¹

17 Apr 2014-Frontiers in Microbiology (Frontiers)-Vol. 5, pp 172-172

TL;DR: The different approaches for the synthesis of recombinant proteins in E. coli are reviewed and recent progress in this ever-growing field is discussed.

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Abstract: Escherichia coli is the organism of choice for the production of recombinant proteins. Its use as a cell factory is well-established and it has become the most popular expression platform. For this reason, there are many molecular tools and protocols at hand for the high-level production of recombinant proteins, such as a vast catalog of expression plasmids, a great number of engineered strains and many cultivation strategies. We review the different approaches for the synthesis of recombinant proteins in E. coli and discuss recent progress in this ever-growing field.

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Journal Article•DOI•

Complete genome sequence of DSM 30083(T), the type strain (U5/41(T)) of Escherichia coli, and a proposal for delineating subspecies in microbial taxonomy.

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Jan P. Meier-Kolthoff¹, Richard L. Hahnke¹, Jörn Petersen¹, Carmen Scheuner¹, Victoria Michael¹, Anne Fiebig¹, Christine Rohde¹, Manfred Rohde, Berthold Fartmann, Lynne Goodwin², Olga Chertkov², T. B. K. Reddy², Amrita Pati², Natalia Ivanova², Victor Markowitz², Nikos C. Kyrpides², Nikos C. Kyrpides³, Tanja Woyke², Markus Göker¹, Hans-Peter Klenk¹ - Show less +16 more•Institutions (3)

Leibniz Association¹, Joint Genome Institute², King Abdulaziz University³

08 Dec 2014-Standards in Genomic Sciences

TL;DR: Analyses of the genome sequences of a large number of E. coli strains and of strains from > 100 other bacterial genera indicate a value of 79-80% dDDH as the most promising threshold for delineating subspecies, which in turn suggests the presence of five subspecies withinE.

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Abstract: Although Escherichia coli is the most widely studied bacterial model organism and often considered to be the model bacterium per se, its type strain was until now forgotten from microbial genomics. As a part of the G enomic E ncyclopedia of B acteria and A rchaea project, we here describe the features of E. coli DSM 30083T together with its genome sequence and annotation as well as novel aspects of its phenotype. The 5,038,133 bp containing genome sequence includes 4,762 protein-coding genes and 175 RNA genes as well as a single plasmid. Affiliation of a set of 250 genome-sequenced E. coli strains, Shigella and outgroup strains to the type strain of E. coli was investigated using digital DNA:DNA-hybridization (dDDH) similarities and differences in genomic G+C content. As in the majority of previous studies, results show Shigella spp. embedded within E. coli and in most cases forming a single subgroup of it. Phylogenomic trees also recover the proposed E. coli phylotypes as monophyla with minor exceptions and place DSM 30083T in phylotype B2 with E. coli S88 as its closest neighbor. The widely used lab strain K-12 is not only genomically but also physiologically strongly different from the type strain. The phylotypes do not express a uniform level of character divergence as measured using dDDH, however, thus an alternative arrangement is proposed and discussed in the context of bacterial subspecies. Analyses of the genome sequences of a large number of E. coli strains and of strains from > 100 other bacterial genera indicate a value of 79-80% dDDH as the most promising threshold for delineating subspecies, which in turn suggests the presence of five subspecies within E. coli.

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367 citations

Cites background from "Recombinant protein expression in E..."

...coli such as metabolic engineering for the production of chemicals and biofuels [14,15], recombinant protein expression [16], the process of binary fission [17], DNA replication and segregation [18], small RNA regulators [19], genetics of the capsular machinery gene cluster [20], as well as comparative genomics [21] and the current status and the progress in clinically relevant E....
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Journal Article•DOI•

NanoLuc: A Small Luciferase Is Brightening Up the Field of Bioluminescence.

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Christopher G. England¹, Emily B. Ehlerding¹, Weibo Cai¹•Institutions (1)

University of Wisconsin-Madison¹

19 Apr 2016-Bioconjugate Chemistry

TL;DR: This review will present the NLuc technology to the scientific community in a nonbiased manner, allowing the audience to adopt their own views of this novel system.

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328 citations

Journal Article•DOI•

Design of virus-based nanomaterials for medicine, biotechnology, and energy

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Amy M. Wen¹, Nicole F. Steinmetz•Institutions (1)

Case Western Reserve University¹

25 Jul 2016-Chemical Society Reviews

TL;DR: This review provides an overview of recent developments in "chemical virology", and surveys the broad distribution of viruses and various methods for producing virus-based nanoparticles, as well as engineering principles used to impart new functionalities.

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Abstract: This review provides an overview of recent developments in “chemical virology.” Viruses, as materials, provide unique nanoscale scaffolds that have relevance in chemical biology and nanotechnology, with diverse areas of applications. Some fundamental advantages of viruses, compared to synthetically programmed materials, include the highly precise spatial arrangement of their subunits into a diverse array of shapes and sizes and many available avenues for easy and reproducible modification. Here, we will first survey the broad distribution of viruses and various methods for producing virus-based nanoparticles, as well as engineering principles used to impart new functionalities. We will then examine the broad range of applications and implications of virus-based materials, focusing on the medical, biotechnology, and energy sectors. We anticipate that this field will continue to evolve and grow, with exciting new possibilities stemming from advancements in the rational design of virus-based nanomaterials.

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293 citations

Journal Article•DOI•

A Review of the Microbial Production of Bioactive Natural Products and Biologics.

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Janette V. Pham¹, Mariamawit A. Yilma¹, Adriana Feliz¹, Murtadha T. Majid¹, Nicholas Maffetone¹, Jorge R. Walker¹, Eunji Kim², Hyo Je Cho³, Jared M. Reynolds¹, Myoung Chong Song², Sung Ryeol Park¹, Sung Ryeol Park⁴, Yeo Joon Yoon² - Show less +9 more•Institutions (4)

The Commonwealth Medical College¹, Ewha Womans University², Kyungpook National University³, Discovery Institute⁴

20 Jun 2019-Frontiers in Microbiology

TL;DR: The structures and diverse biological activities of natural products and recombinant proteins that have been exploited as valuable molecules in medicine, agriculture and insect control are introduced and suggested to inspire the development of new therapeutic agents in academia and industry.

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Abstract: A variety of organisms, such as bacteria, fungi, and plants, produce secondary metabolites, also known as natural products. Natural products have been a prolific source and an inspiration for numerous medical agents with widely divergent chemical structures and biological activities, including antimicrobial, immunosuppressive, anticancer, and anti-inflammatory activities, many of which have been developed as treatments and have potential therapeutic applications for human diseases. Aside from natural products, the recent development of recombinant DNA technology has sparked the development of a wide array of biopharmaceutical products, such as recombinant proteins, offering significant advances in treating a broad spectrum of medical illnesses and conditions. Herein, we will introduce the structures and diverse biological activities of natural products and recombinant proteins that have been exploited as valuable molecules in medicine, agriculture and insect control. In addition, we will explore past and ongoing efforts along with achievements in the development of robust and promising microorganisms as cell factories to produce biologically active molecules. Furthermore, we will review multi-disciplinary and comprehensive engineering approaches directed at improving yields of microbial production of natural products and proteins and generating novel molecules. Throughout this article, we will suggest ways in which microbial-derived biologically active molecular entities and their analogs could continue to inspire the development of new therapeutic agents in academia and industry.

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283 citations

Journal Article•DOI•

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development.

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Nagesh K. Tripathi¹, Ambuj Shrivastava¹•Institutions (1)

Defence Research and Development Establishment¹

20 Dec 2019-Frontiers in Bioengineering and Biotechnology

TL;DR: Recently, advances have been made in the various areas of bioprocessing and are being utilized to develop effective processes for producing recombinant proteins that can be used as therapeutics, vaccines, and diagnostic reagents.

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Abstract: Infectious diseases, along with cancers, are among the main causes of death among humans worldwide. The production of therapeutic proteins for treating diseases at large scale for millions of individuals is one of the essential needs of mankind. Recent progress in the area of recombinant DNA technologies has paved the way to producing recombinant proteins that can be used as therapeutics, vaccines, and diagnostic reagents. Recombinant proteins for these applications are mainly produced using prokaryotic and eukaryotic expression host systems such as mammalian cells, bacteria, yeast, insect cells, and transgenic plants at laboratory scale as well as in large-scale settings. The development of efficient bioprocessing strategies is crucial for industrial production of recombinant proteins of therapeutic and prophylactic importance. Recently, advances have been made in the various areas of bioprocessing and are being utilized to develop effective processes for producing recombinant proteins. These include the use of high-throughput devices for effective bioprocess optimization and of disposable systems, continuous upstream processing, continuous chromatography, integrated continuous bioprocessing, Quality by Design, and process analytical technologies to achieve quality product with higher yield. This review summarizes recent developments in the bioprocessing of recombinant proteins, including in various expression systems, bioprocess development, and the upstream and downstream processing of recombinant proteins.

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249 citations

Cites background from "Recombinant protein expression in E..."

...Codon optimization increases the expression of recombinant protein by many folds (Rosano and Ceccarelli, 2014; Gupta S. K. et al., 2019; Rosano et al., 2019)....
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References

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Open Access

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Journal Article•DOI•

Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes

[...]

F W Studier¹, B A Moffatt¹•Institutions (1)

Brookhaven National Laboratory¹

05 May 1986-Journal of Molecular Biology

TL;DR: A gene expression system based on bacteriophage T7 RNA polymerase has been developed and high levels of accumulation suggest that the RNAs are relatively stable, perhaps in part because their great length and/or stem-and-loop structures at their 3' ends help to protect them against exonucleolytic degradation.

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6,415 citations

"Recombinant protein expression in E..." refers background in this paper

...BL21 was described by Studier in 1986 after various modifications of the B line (Studier and Moffatt, 1986), which in turn Daegelen et al. (2009) traced back to d’Herelle....
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...This highly active polymerase should be provided in another plasmid or, most commonly, it is placed in the bacterial genome in a prophage (λDE3) encoding for the T7 RNAP under the transcriptional control of a lacUV5 promoter (Studier and Moffatt, 1986)....
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Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase.

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D B Smith¹, Kevin S. Johnson²•Institutions (2)

Walter and Eliza Hall Institute of Medical Research¹, University of Melbourne²

15 Jul 1988-Gene

TL;DR: Plasmid expression vectors have been constructed that direct the synthesis of foreign polypeptides in Escherichia coli as fusions with the C terminus of Sj26, a 26-kDa glutathione S-transferase (GST; EC 2.5.1.18) encoded by the parasitic helminth Schistosoma japonicum.

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6,003 citations

"Recombinant protein expression in E..." refers background in this paper

...…popular fusion tags are the maltose-binding protein (MBP; Kapust and Waugh, 1999), N-utilization substance protein A (NusA; Davis et al., 1999), thioredoxin (Trx; LaVallie et al., 1993), glutathione S-transferase (GST; Smith and Johnson, 1988), ubiquitin (Baker, 1996) and SUMO (Butt et al., 2005)....
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...While this promising technology has been proved successful in large-scale fermentors (Voss and Steinbuchel, 2006; Peubez et al., 2010), expression systems based on plasmid addiction are still not widely distributed....
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Protein production by auto-induction in high-density shaking cultures

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F. William Studier¹•Institutions (1)

Brookhaven National Laboratory¹

01 May 2005-Protein Expression and Purification

TL;DR: Investigation of factors that affect stability, growth, and induction of T7 expression strains in shaking vessels led to the recognition that sporadic, unintended induction of expression in complex media, previously reported by others, is almost certainly caused by small amounts of lactose.

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5,395 citations

"Recombinant protein expression in E..." refers background in this paper

...A tighter control can be achieved by the addition of 0.2–1% w/v glucose in the medium as rich media prepared with tryptone or peptone may contain the inducer lactose (Studier, 2005)....
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...In that report, the concept of autoinduction was developed (Studier, 2005)....
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...…poses a problem, the media can be buffered with phosphate salts at 50 mM. 2xYT, TB (Terrific Broth) and SB (Super Broth) media recipes are available elsewhere and have been shown to be superior to LB for reaching higher cell densities (Madurawe et al., 2000; Atlas, 2004; Studier, 2005)....
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...Not surprisingly, increasing the amount of peptone or yeast extract leads to higher cell densities (Studier, 2005)....
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Journal Article•DOI•

Construction and characterization of new cloning vehicles. II. A multipurpose cloning system.

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Francisco Bolívar¹, Raymond L. Rodriguez, Patricia J. Greene¹, Mary C. Betlach, Herbert L. Heyneker, Herbert W. Boyer, Jorge H. Crosa, Stanley Falkow - Show less +4 more•Institutions (1)

University of California, San Francisco¹

01 Nov 1977-Gene

TL;DR: In vitro recombination techniques were used to construct a new cloning vehicle, pBR322, which is a relaxed replicating plasmid, does not produce and is sensitive to colicin E1, and carries resistance genes to the antibiotics ampicillin (Ap) and tetracycline (Tc).

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5,235 citations

"Recombinant protein expression in E..." refers background in this paper

...Commonly used vectors, such as the pET series, possess the pMB1 origin (ColE1-derivative, 15–60 copies per cell; Bolivar et al., 1977) while a mutated version of the pMB1 origin is present in the pUC series (500–700 copies per cell; Minton, 1984)....
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Journal Article•DOI•

Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter.

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Luz-Maria Guzman¹, Dominique Belin¹, M. J. Carson¹, Jon Beckwith¹•Institutions (1)

Harvard University¹

01 Jul 1995-Journal of Bacteriology

TL;DR: The tight regulation of the PBAD promoter is exploited to study the phenotypes of null mutations of essential genes and the use of pBAD vectors as an expression system is explored.

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Abstract: We have constructed a series of plasmid vectors (pBAD vectors) containing the PBAD promoter of the araBAD (arabinose) operon and the gene encoding the positive and negative regulator of this promoter, araC. Using the phoA gene and phoA fusions to monitor expression in these vectors, we show that the ratio of induction/repression can be 1,200-fold, compared with 50-fold for PTAC-based vectors. phoA expression can be modulated over a wide range of inducer (arabinose) concentrations and reduced to extremely low levels by the presence of glucose, which represses expression. Also, the kinetics of induction and repression are very rapid and significantly affected by the ara allele in the host strain. Thus, the use of this system which can be efficiently and rapidly turned on and off allows the study of important aspects of bacterial physiology in a very simple manner and without changes of temperature. We have exploited the tight regulation of the PBAD promoter to study the phenotypes of null mutations of essential genes and explored the use of pBAD vectors as an expression system.

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4,997 citations

"Recombinant protein expression in E..." refers background or methods in this paper

...Frontiers in Microbiology | Microbiotechnology, Ecotoxicology and Bioremediation April 2014 | Volume 5 | Article 172 | 2...
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...Moreover, the field is always expanding and even after almost 40 years from the first human protein obtained in E. coli (Itakura et al., 1977), there is still much room for improvement....
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...For the dual expression of recombinant proteins using two plasmids, systems with the p15A ori are available (pACYC and pBAD series of plasmids, 10–12 copies per cell; Chang and Cohen, 1978; Guzman et al., 1995)....
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...This is the case of the araPBAD promoter present in the pBAD vectors (Guzman et al., 1995)....
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...However, exponential growth in www.frontiersin.org April 2014 | Volume 5 | Article 172 | 1 complex media leads to densities nowhere near that number....
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