Recombinant protein expression in microbial systems

856.1K

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28

authors

10

articles

Editorial

08 July 2014

Recombinant protein expression in microbial systems

Germán L. Rosano

and

Eduardo A. Ceccarelli

21,205 views

74 citations

Editors

2

Eduardo A. Ceccarelli

CONICET Instituto de Biología Molecular y Celular de Rosario (IBR)

Germán Leandro Rosano

CONICET Instituto de Biología Molecular y Celular de Rosario (IBR)

Impact

Anatomy of an expression vector. The figure depicts the major features present in common expression vectors. All of them are described in the text. The affinity tags and coding sequences for their removal were positioned arbitrarily at the N-terminus for simplicity. MCS, multiple cloning site. Striped patterned box: coding sequence for the desired protein.

Review

17 April 2014

Recombinant protein expression in Escherichia coli: advances and challenges

Germán L. Rosano

and

Eduardo A. Ceccarelli

Escherichia coli is one of the organisms 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 heterologous 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.

591,336 views

2,010 citations

Strategies for soluble protein production in E. coli. (A) Expression vectors should be carefully selected in order to incorporate specific features that affect the protein production in E. coli such as solubility and/or affinity fusion tags, and to direct the protein synthesis to the E. coli cytoplasm or periplasm. Other important features include: the replicon, antibiotic-resistance markers, and transcriptional promoters (Jana and Deb, 2005; Sorensen and Mortensen, 2005a). (B) The optimization of expression conditions often directs the soluble protein production in E. coli, and it relies on a trial-and-error basis: to get a soluble TP, it may require the selection and testing of several engineered E. coli strains and cultivation conditions, and sometimes the initial expression vector has also to be re-designed. (C) The protein purification strategy should already be defined at the beginning when selecting the expression vector: if an affinity tag is incorporated, then a first affinity chromatography step should be conducted. On the other hand, if an affinity tag is prohibit, other strategies, namely, ion exchange, size exclusion, or hydrophobic interaction chromatography should be tested. After the first purification step, the TP may or may not be sufficiently pure. When it is not pure, further purification steps with other chromatographic strategies need to be conducted. (D–E) The protein quality is an essential requirement for many structural and functional application studies: a purified soluble protein may be aggregated, without a defined secondary structure, and it may also present a low thermal stability. Therefore, a biophysical characterization is often required before proceeding to the final protein’s application.

Review

19 February 2014

Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: the novel Fh8 system

Sofia Costa

, 2 more and

Lucília Domingues

115,475 views

372 citations

Review

27 February 2014

Making recombinant proteins in filamentous fungi- are we expecting too much?

21,763 views

126 citations

Mini Review

17 February 2014

Algae-based oral recombinant vaccines

Elizabeth A. Specht

and

Stephen P. Mayfield

19,592 views

120 citations

Perspective

05 March 2014

Playing catch-up with Escherichia coli: using yeast to increase success rates in recombinant protein production experiments

Roslyn M. Bill

20,767 views

94 citations

Original Research

25 February 2014

Generation of a vector suite for protein solubility screening

Agustín Correa

, 4 more and

Pablo Oppezzo

9,472 views

26 citations

Mini Review

14 February 2014

Inclusion bodies: not that bad…

Ana Ramón

, 1 more and

Mónica Marín

24,108 views

98 citations

Review

06 February 2014

Flavoprotein monooxygenases for oxidative biocatalysis: recombinant expression in microbial hosts and applications

Romina D. Ceccoli

, 1 more and

Daniela V. Rial

9,189 views

47 citations

Schematic representation of a synthetic biology network applied to the design of novel bio-based parts and devices, as well as to the engineering of existing natural biological systems for the development of target products.

Mini Review

04 February 2014

Expression of codon optimized genes in microbial systems: current industrial applications and perspectives

Claudia Elena

, 3 more and

Hugo G. Menzella

16,857 views

109 citations

Open for submission

Frontiers in Microbiology

The Role Of Microbial Communities In Environmental Engineering Systems

Edited by Yuepeng Sun, Dunzhu Li, Bo Li, Keli Zhao

Deadline

30 April 2025

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About

With the advent of recombinant DNA technology, expressing heterologous proteins in microorganisms rapidly became the method of choice for their production at laboratory and industrial scale. Bacteria, yeasts and other hosts can be grown to high biomass levels efficiently and inexpensively. Obtaining high yields of recombinant proteins from this material was only feasible thanks to constant research on microbial genetics and physiology that led to novel strains, plasmids and cultivation strategies.
Despite the spectacular expansion of the field, there is still much room for progress. Improving the levels of expression and the solubility of a recombinant protein can be quite challenging. Accumulation of the product in the cell can lead to stress responses which affect cell growth. Buildup of insoluble and biologically inactive aggregates (inclusion bodies) lowers the yield of production. This is particularly true for obtaining membrane proteins or high-molecular weight and multi-domain proteins. Also, obtaining eukaryotic proteins in a prokaryotic background (for example, plant or animal proteins in bacteria) results in a product that lack post-translational modifications, often required for functionality. Changing to a eukaryotic host (yeasts or filamentous fungi) may not be a proper solution since the pattern of sugar modifications is different than in higher eukaryotes.
Still, many advances in the last couple of decades have provided to researchers a wide variety of strategies to maximize the production of their recombinant protein of choice. Everything starts with the careful selection of the host. Be it bacteria or yeast, a broad list of strains is available for overcoming codon use bias, incorrect disulfide bond formation, protein toxicity and lack of post-translational modifications. Also, a huge catalog of plasmids allows choosing for different fusion partners for improving solubility, protein secretion, chaperone co-expression, antibiotic resistance and promoter strength. Next, controlling culture conditions like temperature, inducer and media composition can bolster recombinant protein production.
With this Research Topic, we aim to provide an encyclopedic account of the existing approaches to the expression of recombinant proteins in microorganisms, highlight recent discoveries and analyze the future prospects of this exciting and ever-growing field.

With the advent of recombinant DNA technology, expressing heterologous proteins in microorganisms rapidly became the method of choice for their production at laboratory and industrial scale. Bacteria, yeasts and other hosts can be grown to high biomass levels efficiently and inexpensively. Obtaining high yields of recombinant proteins from this material was only feasible thanks to constant research on microbial genetics and physiology that led to novel strains, plasmids and cultivation strategies.
Despite the spectacular expansion of the field, there is still much room for progress. Improving the levels of expression and the solubility of a recombinant protein can be quite challenging. Accumulation of the product in the cell can lead to stress responses which affect cell growth. Buildup of insoluble and biologically inactive aggregates (inclusion bodies) lowers the yield of production. This is particularly true for obtaining membrane proteins or high-molecular weight and multi-domain proteins. Also, obtaining eukaryotic proteins in a prokaryotic background (for example, plant or animal proteins in bacteria) results in a product that lack post-translational modifications, often required for functionality. Changing to a eukaryotic host (yeasts or filamentous fungi) may not be a proper solution since the pattern of sugar modifications is different than in higher eukaryotes.
Still, many advances in the last couple of decades have provided to researchers a wide variety of strategies to maximize the production of their recombinant protein of choice. Everything starts with the careful selection of the host. Be it bacteria or yeast, a broad list of strains is available for overcoming codon use bias, incorrect disulfide bond formation, protein toxicity and lack of post-translational modifications. Also, a huge catalog of plasmids allows choosing for different fusion partners for improving solubility, protein secretion, chaperone co-expression, antibiotic resistance and promoter strength. Next, controlling culture conditions like temperature, inducer and media composition can bolster recombinant protein production.
With this Research Topic, we aim to provide an encyclopedic account of the existing approaches to the expression of recombinant proteins in microorganisms, highlight recent discoveries and analyze the future prospects of this exciting and ever-growing field.

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Editors

Eduardo A. Ceccarelli

CONICET Instituto de Biología Molecular y Celular de Rosario (IBR)

Germán Leandro Rosano

CONICET Instituto de Biología Molecular y Celular de Rosario (IBR)

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856,138 Total views

713,558 Article views

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Microbiotechnology

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Microbiotechnology

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