Special Issue "Recombinant Protein Expression in Microorganisms"

A special issue of Microorganisms (ISSN 2076-2607). This special issue belongs to the section "Microbial Biotechnology".

Deadline for manuscript submissions: closed (20 December 2018)

Special Issue Editors

Guest Editor
Prof. Dr. Trygve Brautaset

Department of Biotechnology, Norwegian University of Science and Technology, Trondheim, Norway
Website | E-Mail
Phone: +47 98 28 39 77
Interests: Microbial molecular biology, synthetic biology, systems biology, recombinant expression, methylotrophy, digital biotechnology
Co-Guest Editor
Prof. Dr. Svein Valla *

Department of Biotechnology, Norwegian University of Science and Technology, Trondheim, Norway
Website | E-Mail
Interests: Molecular biology, microbiology, biopolymers, systems biology, recombinant expression, bioremediation
* Deceased, September 2017

Special Issue Information

Dear Colleagues,

Recombinant protein expression is a fundamental discipline in molecular biology, as well as a key technology to produce any specific protein for scientific and applied purposes. Today, virtually any protein from any origin can be recombinantly overproduced, and microorganisms are in many instances the preferred production hosts. Recombinant expression has been subject for intensive research for decades and yet still today there are fundamental aspects of this science we do not understand. Presumably, critical information is hidden in the gene coding sequences, beyond well-understood rules concerning rare codons and secondary structure formation at the mRNA level. Challenges can be at any level, including host-toxicity, product insolubility and non-functionality, poor translation, and lack of necessary post-translational modification. Genetic tools, superior hosts and new software are being rapidly developed for improved design, by taking all possible parameters (gene coding sequences, promoters, untranslated leaders regions, transcriptional terminators, mRNA stabilities, protein folding, translational efficiencies) into consideration. Still today, recombinant protein expression is largely a matter of trial and error.

In this Special issue of Microorganisms, we invite you to send contributions concerning any aspects related to recombinant protein expression in microorganisms, including genetic tools developments, bioinformatics tools for better predictions of expression levels and protein solubility, methodologies for product detection and characterization, as well as systems biology driven and laboratory-evolution based strategies to better understand and create novel host strains (both eukaryotic and prokaryotic) for improved recombinant protein production. 

Prof. Dr. Trygve Brautaset
Prof. Dr. Svein Valla
Guest Editors

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Keywords

  • Microbial cell factories
  • Expression systems
  • Protein folding
  • Bioinformatics
  • Modeling
  • Synthetic biology
  • Systems biology
  • Genetic engineering
  • Biotechnology

Published Papers (13 papers)

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Research

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Open AccessArticle ATP- and Polyphosphate-Dependent Glucokinases from Aerobic Methanotrophs
Microorganisms 2019, 7(2), 52; https://doi.org/10.3390/microorganisms7020052
Received: 26 December 2018 / Revised: 1 February 2019 / Accepted: 12 February 2019 / Published: 14 February 2019
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Abstract
The genes encoding adenosine triphosphate (ATP)- and polyphosphate (polyP)-dependent glucokinases (Glk) were identified in the aerobic obligate methanotroph Methylomonas sp. 12. The recombinant proteins were obtained by the heterologous expression of the glk genes in Esherichia coli. ATP-Glk behaved as a multimeric [...] Read more.
The genes encoding adenosine triphosphate (ATP)- and polyphosphate (polyP)-dependent glucokinases (Glk) were identified in the aerobic obligate methanotroph Methylomonas sp. 12. The recombinant proteins were obtained by the heterologous expression of the glk genes in Esherichia coli. ATP-Glk behaved as a multimeric protein consisting of di-, tri-, tetra-, penta- and hexamers with a subunit molecular mass of 35.5 kDa. ATP-Glk phosphorylated glucose and glucosamine using ATP (100% activity), uridine triphosphate (UTP) (85%) or guanosine triphosphate (GTP) (71%) as a phosphoryl donor and exhibited the highest activity in the presence of 5 mM Mg2+ at pH 7.5 and 65 °C but was fully inactivated after a short-term incubation at this temperature. According to a gel filtration in the presence of polyP, the polyP-dependent Glk was a dimeric protein (2 × 28 kDa). PolyP-Glk phosphorylated glucose, mannose, 2-deoxy-D-glucose, glucosamine and N-acetylglucosamine using polyP as the phosphoryl donor but not using nucleoside triphosphates. The Km values of ATP-Glk for glucose and ATP were about 78 μM, and the Km values of polyP-Glk for glucose and polyP(n=45) were 450 and 21 μM, respectively. The genomic analysis of methanotrophs showed that ATP-dependent glucokinase is present in all sequenced methanotrophs, with the exception of the genera Methylosinus and Methylocystis, whereas polyP-Glks were found in all species of the genus Methylomonas and in Methylomarinum vadi only. This work presents the first characterization of polyphosphate specific glucokinase in a methanotrophic bacterium. Full article
(This article belongs to the Special Issue Recombinant Protein Expression in Microorganisms)
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Open AccessArticle An Alternative Platform for Protein Expression Using an Innate Whole Expression Module from Metagenomic DNA
Microorganisms 2019, 7(1), 9; https://doi.org/10.3390/microorganisms7010009
Received: 8 November 2018 / Revised: 20 December 2018 / Accepted: 3 January 2019 / Published: 8 January 2019
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Abstract
Many integrated gene clusters beyond a single genetic element are commonly trapped as the result of promoter traps in (meta)genomic DNA libraries. Generally, a single element, which is mainly the promoter, is deduced from the resulting gene clusters and employed to construct a [...] Read more.
Many integrated gene clusters beyond a single genetic element are commonly trapped as the result of promoter traps in (meta)genomic DNA libraries. Generally, a single element, which is mainly the promoter, is deduced from the resulting gene clusters and employed to construct a new expression vector. However, expression patterns of target proteins under the incorporated promoter are often inconsistent with those shown in clones harboring plasmids with gene clusters. These results suggest that the integrated set of gene clusters with diverse cis- and trans-acting elements is evolutionarily tuned as a complete set for gene expression, and is an expression module with all the components for the expression of a nested open reading frame (ORF). This possibility is further supported by truncation and/or serial deletion analysis of this module in which the expression of the nested ORF is highly fluctuated or reduced frequently, despite being supported by plentiful cis-acting elements in the spanning regions around the ORF such as the promoter, ribosome binding site (RBS), terminator, and 3′-/5′-UTRs for gene expression. Here, we examined whether an innate module with a naturally overexpressed gene could be considered as a scaffold for an expression system. For a proof-of-principle study, we mined a complete expression module with an innately overexpressed ORF in E. coli from a metagenomics DNA library, and incorporated it into a vector that had no regulatory element for expressing the insert. We obtained successful expression of several inserts such as MBP, GFPuv, β-glucosidase, and esterase using this simple construct without tuning and codon optimization of the target insert. Full article
(This article belongs to the Special Issue Recombinant Protein Expression in Microorganisms)
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Open AccessArticle Expression and Purification of Chemokine MIP-3α (CCL20) through a Calmodulin-Fusion Protein System
Microorganisms 2019, 7(1), 8; https://doi.org/10.3390/microorganisms7010008
Received: 30 November 2018 / Revised: 22 December 2018 / Accepted: 2 January 2019 / Published: 8 January 2019
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Abstract
Human macrophage inflammatory protein 3α (MIP-3α), also known as CCL20, is a 70 amino acid chemokine that selectively binds and activates chemokine receptor 6 (CCR6). This chemokine is responsible for inducing the migration of immature dendritic cells, effector, or memory T-cells, and B-cells. [...] Read more.
Human macrophage inflammatory protein 3α (MIP-3α), also known as CCL20, is a 70 amino acid chemokine that selectively binds and activates chemokine receptor 6 (CCR6). This chemokine is responsible for inducing the migration of immature dendritic cells, effector, or memory T-cells, and B-cells. Moreover, the MIP-3α protein has been shown to display direct antimicrobial, antiviral and antiprotozoal activities. Because of the potential therapeutic uses of this protein, the efficient production of MIP-3α is of great interest. However, bacterial recombinant production of the MIP-3α protein has been limited by the toxicity of this extremely basic protein (pI 9.7) toward prokaryotic cells, and by solubility problems during expression and purification. In an attempt to overcome these issues, we have investigated the bacterial recombinant expression of MIP-3α by using several common expression and fusion tags, including 6× histidine (His), small ubiquitin modifier protein (SUMO), thioredoxin (TRX), ketosteroid isomerase (KSI), and maltose binding protein (MBP). We have also evaluated a recently introduced calmodulin (CaM)-tag that has been used for the effective expression of many basic antimicrobial peptides (AMPs). Here, we show that the CaM fusion tag system effectively expressed soluble MIP-3α in the cytoplasm of Escherichia coli with good yields. Rapid purification was facilitated by the His-tag that was integrated in the CaM-fusion protein system. Multidimensional nuclear magnetic resonance (NMR) studies demonstrated that the recombinant protein was properly folded, with the correct formation of disulfide bonds. In addition, the recombinant MIP-3α had antibacterial activity, and was shown to inhibit the formation of Pseudomonas aeruginosa biofilms. Full article
(This article belongs to the Special Issue Recombinant Protein Expression in Microorganisms)
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Open AccessArticle Inclusion Body Bead Size in E. coli Controlled by Physiological Feeding
Microorganisms 2018, 6(4), 116; https://doi.org/10.3390/microorganisms6040116
Received: 1 October 2018 / Revised: 16 November 2018 / Accepted: 22 November 2018 / Published: 25 November 2018
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Abstract
The Gram-negative bacterium E. coli is the host of choice for producing a multitude of recombinant proteins relevant in the pharmaceutical industry. Generally, cultivation is easy, media are cheap, and a high product titer can be obtained. However, harsh induction procedures combined with [...] Read more.
The Gram-negative bacterium E. coli is the host of choice for producing a multitude of recombinant proteins relevant in the pharmaceutical industry. Generally, cultivation is easy, media are cheap, and a high product titer can be obtained. However, harsh induction procedures combined with the usage of IPTG (isopropyl β-d-1 thiogalactopyranoside) as an inducer are often believed to cause stress reactions, leading to intracellular protein aggregates, which are so known as so-called inclusion bodies (IBs). Downstream applications in bacterial processes cause the bottleneck in overall process performance, as bacteria lack many post-translational modifications, resulting in time and cost-intensive approaches. Especially purification of inclusion bodies is notoriously known for its long processing times and low yields. In this contribution, we present screening strategies for determination of inclusion body bead size in an E. coli-based bioprocess producing exclusively inclusion bodies. Size can be seen as a critical quality attribute (CQA), as changes in inclusion body behavior have a major effect on subsequent downstream processing. A model-based approach was used, aiming to trigger a distinct inclusion body size: Physiological feeding control, using qs,C as a critical process parameter, has a high impact on inclusion body size and could be modelled using a hyperbolic saturation mechanism calculated in form of a cumulated substrate uptake rate. Within this model, the sugar uptake rate of the cells, in the form of the cumulated sugar uptake-value, was simulated and considered being a key performance indicator for determination of the desired size. We want to highlight that the usage of the mentioned screening strategy in combination with a model-based approach will allow tuning of the process towards a certain inclusion body size using a qs based control only. Optimized inclusion body size at the time-point of harvest should stabilize downstream processing and, therefore, increase the overall time-space yield. Furthermore, production of distinct inclusion body size may be interesting for application as a biocatalyst and nanoparticulate material. Full article
(This article belongs to the Special Issue Recombinant Protein Expression in Microorganisms)
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Open AccessArticle Automated Cell Treatment for Competence and Transformation of Escherichia coli in a High-Throughput Quasi-Turbidostat Using Microtiter Plates
Microorganisms 2018, 6(3), 60; https://doi.org/10.3390/microorganisms6030060
Received: 5 May 2018 / Revised: 10 June 2018 / Accepted: 22 June 2018 / Published: 25 June 2018
Cited by 1 | PDF Full-text (3715 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Metabolic engineering and genome editing strategies often lead to large strain libraries of a bacterial host. Nevertheless, the generation of competent cells is the basis for transformation and subsequent screening of these strains. While preparation of competent cells is a standard procedure in [...] Read more.
Metabolic engineering and genome editing strategies often lead to large strain libraries of a bacterial host. Nevertheless, the generation of competent cells is the basis for transformation and subsequent screening of these strains. While preparation of competent cells is a standard procedure in flask cultivations, parallelization becomes a challenging task when working with larger libraries and liquid handling stations as transformation efficiency depends on a distinct physiological state of the cells. We present a robust method for the preparation of competent cells and their transformation. The strength of the method is that all cells on the plate can be maintained at a high growth rate until all cultures have reached a defined cell density regardless of growth rate and lag phase variabilities. This allows sufficient transformation in automated high throughput facilities and solves important scheduling issues in wet-lab library screenings. We address the problem of different growth rates, lag phases, and initial cell densities inspired by the characteristics of continuous cultures. The method functions on a fully automated liquid handling platform including all steps from the inoculation of the liquid cultures to plating and incubation on agar plates. The key advantage of the developed method is that it enables cell harvest in 96 well plates at a predefined time by keeping fast growing cells in the exponential phase as in turbidostat cultivations. This is done by a periodic monitoring of cell growth and a controlled dilution specific for each well. With the described methodology, we were able to transform different strains in parallel. The transformants produced can be picked and used in further automated screening experiments. This method offers the possibility to transform any combination of strain- and plasmid library in an automated high-throughput system, overcoming an important bottleneck in the high-throughput screening and the overall chain of bioprocess development. Full article
(This article belongs to the Special Issue Recombinant Protein Expression in Microorganisms)
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Open AccessArticle Development of Versatile Vectors for Heterologous Expression in Bacillus
Microorganisms 2018, 6(2), 51; https://doi.org/10.3390/microorganisms6020051
Received: 28 March 2018 / Revised: 1 June 2018 / Accepted: 5 June 2018 / Published: 7 June 2018
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Abstract
The discovery of new enzymes for industrial application relies on a robust discovery pipeline. Such a pipeline should facilitate efficient molecular cloning, recombinant expression and functional screening procedures. Previously, we have developed a vector set for heterologous expression in Escherichia coli. Here, [...] Read more.
The discovery of new enzymes for industrial application relies on a robust discovery pipeline. Such a pipeline should facilitate efficient molecular cloning, recombinant expression and functional screening procedures. Previously, we have developed a vector set for heterologous expression in Escherichia coli. Here, we supplement the catalogue with vectors for expression in Bacillus. The vectors are made compatible with a versatile cloning procedure based on type IIS restriction enzymes and T4 DNA ligase, and encompass an effective counter-selection procedure and complement the set of vectors with options for secreted expression. We validate the system with expression of recombinant subtilisins, which are generally challenging to express in a heterologous system. The complementarity of the E. coli and Bacillus systems allows rapid switching between the two commonly used hosts without comprehensive intermediate cloning steps. The vectors described are not limited to the expression of certain enzymes, but could also be applied for the expression of other enzymes for more generalized enzyme discovery or development. Full article
(This article belongs to the Special Issue Recombinant Protein Expression in Microorganisms)
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Open AccessArticle Comparing the Recombinant Protein Production Potential of Planktonic and Biofilm Cells
Microorganisms 2018, 6(2), 48; https://doi.org/10.3390/microorganisms6020048
Received: 6 April 2018 / Revised: 18 May 2018 / Accepted: 21 May 2018 / Published: 24 May 2018
Cited by 1 | PDF Full-text (1425 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Recombinant protein production in bacterial cells is commonly performed using planktonic cultures. However, the natural state for many bacteria is living in communities attached to surfaces forming biofilms. In this work, a flow cell system was used to compare the production of a [...] Read more.
Recombinant protein production in bacterial cells is commonly performed using planktonic cultures. However, the natural state for many bacteria is living in communities attached to surfaces forming biofilms. In this work, a flow cell system was used to compare the production of a model recombinant protein (enhanced green fluorescent protein, eGFP) between planktonic and biofilm cells. The fluorometric analysis revealed that when the system was in steady state, the average specific eGFP production from Escherichia coli biofilm cells was 10-fold higher than in planktonic cells. Additionally, epifluorescence microscopy was used to determine the percentage of eGFP-expressing cells in both planktonic and biofilm populations. In steady state, the percentage of planktonic-expressing cells oscillated around 5%, whereas for biofilms eGFP-expressing cells represented on average 21% of the total cell population. Therefore, the combination of fluorometric and microscopy data allowed us to conclude that E. coli biofilm cells can have a higher recombinant protein production capacity when compared to their planktonic counterparts. Full article
(This article belongs to the Special Issue Recombinant Protein Expression in Microorganisms)
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Open AccessArticle Recombinant Inga Laurina Trypsin Inhibitor (ILTI) Production in Komagataella Phaffii Confirms Its Potential Anti-Biofilm Effect and Reveals an Anti-Tumoral Activity
Microorganisms 2018, 6(2), 37; https://doi.org/10.3390/microorganisms6020037
Received: 30 March 2018 / Revised: 23 April 2018 / Accepted: 24 April 2018 / Published: 28 April 2018
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Abstract
Protease inhibitors have a broad biotechnological application ranging from medical drugs to anti-microbial agents. The Inga laurina trypsin inhibitor (ILTI) previously showed a great in vitro inhibitory effect under the adherence of Staphylococcus species, being a strong candidate for use as an anti-biofilm [...] Read more.
Protease inhibitors have a broad biotechnological application ranging from medical drugs to anti-microbial agents. The Inga laurina trypsin inhibitor (ILTI) previously showed a great in vitro inhibitory effect under the adherence of Staphylococcus species, being a strong candidate for use as an anti-biofilm agent. Nevertheless, this is found in small quantities in its sources, which impairs its utilization at an industrial scale. Within this context, heterologous production using recombinant microorganisms is one of the best options to scale up the recombinant protein production. Thus, this work aimed at utilizing Komagataella phaffii to produce recombinant ILTI. For this, the vector pPIC9K+ILTI was constructed and inserted into the genome of the yeast K. phaffii, strain GS115. The protein expression was highest after 48 h using methanol 1%. A matrix-assisted laser desorption ionization–time-of-flight (MALDI–TOF) analysis was performed to confirm the production of the recombinant ILTI and its activity was investigated trough inhibitory assays using the synthetic substrate Nα-Benzoyl-D,L-arginine p-nitroanilide hydrochloride (BAPNA). Finally, recombinant ILTI (rILTI) was used in assays, showing that there was no significant difference between native and recombinant ILTI in its inhibitory activity in biofilm formation. Anti-tumor assay against Ehrlich ascites tumor (EAT) cells showed that rILTI has a potential anti-tumoral effect, showing the same effect as Melittin when incubated for 48 h in concentrations above 25 µg/mL. All together the results suggests broad applications for rILTI. Full article
(This article belongs to the Special Issue Recombinant Protein Expression in Microorganisms)
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Review

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Open AccessReview Bioreactor-Scale Strategies for the Production of Recombinant Protein in the Yeast Yarrowia lipolytica
Microorganisms 2019, 7(2), 40; https://doi.org/10.3390/microorganisms7020040
Received: 17 January 2019 / Revised: 28 January 2019 / Accepted: 29 January 2019 / Published: 30 January 2019
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Abstract
Recombinant protein production represents a multibillion-dollar market. Therefore, it constitutes an important research field both in academia and industry. The use of yeast as a cell factory presents several advantages such as ease of genetic manipulation, growth at high cell density, and the [...] Read more.
Recombinant protein production represents a multibillion-dollar market. Therefore, it constitutes an important research field both in academia and industry. The use of yeast as a cell factory presents several advantages such as ease of genetic manipulation, growth at high cell density, and the possibility of post-translational modifications. Yarrowia lipolytica is considered as one of the most attractive hosts due to its ability to metabolize raw substrate, to express genes at a high level, and to secrete protein in large amounts. In recent years, several reviews have been dedicated to genetic tools developed for this purpose. Though the construction of efficient cell factories for recombinant protein synthesis is important, the development of an efficient process for recombinant protein production in a bioreactor constitutes an equally vital aspect. Indeed, a sports car cannot drive fast on a gravel road. The aim of this review is to provide a comprehensive snapshot of process tools to consider for recombinant protein production in bioreactor using Y. lipolytica as a cell factory, in order to facilitate the decision-making for future strain and process engineering. Full article
(This article belongs to the Special Issue Recombinant Protein Expression in Microorganisms)
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Open AccessReview Application of Continuous Culture Methods to Recombinant Protein Production in Microorganisms
Microorganisms 2018, 6(3), 56; https://doi.org/10.3390/microorganisms6030056
Received: 20 April 2018 / Revised: 28 May 2018 / Accepted: 19 June 2018 / Published: 21 June 2018
Cited by 2 | PDF Full-text (239 KB) | HTML Full-text | XML Full-text
Abstract
Depending on the environmental conditions, cells adapt their metabolism and specific growth rate. Rearrangements occur on many different levels such as macromolecular composition, gene and protein expression, morphology and metabolic flux patterns. As the interplay of these processes also determines the output of [...] Read more.
Depending on the environmental conditions, cells adapt their metabolism and specific growth rate. Rearrangements occur on many different levels such as macromolecular composition, gene and protein expression, morphology and metabolic flux patterns. As the interplay of these processes also determines the output of a recombinant protein producing system, having control over specific growth rate of the culture is advantageous. Continuous culture methods were developed to grow cells in a constant environment and have been used for decades to study basic microbial physiology in a controlled and reproducible manner. Our review summarizes the uses of continuous cultures in cell physiology studies and process development, with a focus on recombinant protein-producing microorganisms. Full article
(This article belongs to the Special Issue Recombinant Protein Expression in Microorganisms)
Open AccessReview Polyionic Tags as Enhancers of Protein Solubility in Recombinant Protein Expression
Microorganisms 2018, 6(2), 47; https://doi.org/10.3390/microorganisms6020047
Received: 9 April 2018 / Revised: 16 May 2018 / Accepted: 21 May 2018 / Published: 23 May 2018
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Abstract
Since the introduction of recombinant protein expression in the second half of the 1970s, the growth of the biopharmaceutical field has been rapid and protein therapeutics has come to the foreground. Biophysical and structural characterisation of recombinant proteins is the essential prerequisite for [...] Read more.
Since the introduction of recombinant protein expression in the second half of the 1970s, the growth of the biopharmaceutical field has been rapid and protein therapeutics has come to the foreground. Biophysical and structural characterisation of recombinant proteins is the essential prerequisite for their successful development and commercialisation as therapeutics. Despite the challenges, including low protein solubility and inclusion body formation, prokaryotic host systems and particularly Escherichia coli, remain the system of choice for the initial attempt of production of previously unexpressed proteins. Several different approaches have been adopted, including optimisation of growth conditions, expression in the periplasmic space of the bacterial host or co-expression of molecular chaperones, to assist correct protein folding. A very commonly employed approach is also the use of protein fusion tags that enhance protein solubility. Here, a range of experimentally tested peptide tags, which present specific advantages compared to protein fusion tags and the concluding remarks of these experiments are reviewed. Finally, a concept to design solubility-enhancing peptide tags based on a protein’s pI is suggested. Full article
(This article belongs to the Special Issue Recombinant Protein Expression in Microorganisms)
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Open AccessReview Genetic Tools and Techniques for Recombinant Expression in Thermophilic Bacillaceae
Microorganisms 2018, 6(2), 42; https://doi.org/10.3390/microorganisms6020042
Received: 16 April 2018 / Revised: 2 May 2018 / Accepted: 3 May 2018 / Published: 10 May 2018
Cited by 3 | PDF Full-text (488 KB) | HTML Full-text | XML Full-text
Abstract
Although Escherichia coli and Bacillus subtilis are the most prominent bacterial hosts for recombinant protein production by far, additional species are being explored as alternatives for production of difficult-to-express proteins. In particular, for thermostable proteins, there is a need for hosts able to [...] Read more.
Although Escherichia coli and Bacillus subtilis are the most prominent bacterial hosts for recombinant protein production by far, additional species are being explored as alternatives for production of difficult-to-express proteins. In particular, for thermostable proteins, there is a need for hosts able to properly synthesize, fold, and excrete these in high yields, and thermophilic Bacillaceae represent one potentially interesting group of microorganisms for such purposes. A number of thermophilic Bacillaceae including B. methanolicus, B. coagulans, B. smithii, B. licheniformis, Geobacillus thermoglucosidasius, G. kaustophilus, and G. stearothermophilus are investigated concerning physiology, genomics, genetic tools, and technologies, altogether paving the way for their utilization as hosts for recombinant production of thermostable and other difficult-to-express proteins. Moreover, recent successful deployments of CRISPR/Cas9 in several of these species have accelerated the progress in their metabolic engineering, which should increase their attractiveness for future industrial-scale production of proteins. This review describes the biology of thermophilic Bacillaceae and in particular focuses on genetic tools and methods enabling use of these organisms as hosts for recombinant protein production. Full article
(This article belongs to the Special Issue Recombinant Protein Expression in Microorganisms)
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Open AccessReview Comparison of Yeasts as Hosts for Recombinant Protein Production
Microorganisms 2018, 6(2), 38; https://doi.org/10.3390/microorganisms6020038
Received: 30 March 2018 / Revised: 23 April 2018 / Accepted: 24 April 2018 / Published: 29 April 2018
Cited by 3 | PDF Full-text (1890 KB) | HTML Full-text | XML Full-text
Abstract
Recombinant protein production emerged in the early 1980s with the development of genetic engineering tools, which represented a compelling alternative to protein extraction from natural sources. Over the years, a high level of heterologous protein was made possible in a variety of hosts [...] Read more.
Recombinant protein production emerged in the early 1980s with the development of genetic engineering tools, which represented a compelling alternative to protein extraction from natural sources. Over the years, a high level of heterologous protein was made possible in a variety of hosts ranging from the bacteria Escherichia coli to mammalian cells. Recombinant protein importance is represented by its market size, which reached $1654 million in 2016 and is expected to reach $2850.5 million by 2022. Among the available hosts, yeasts have been used for producing a great variety of proteins applied to chemicals, fuels, food, and pharmaceuticals, being one of the most used hosts for recombinant production nowadays. Historically, Saccharomyces cerevisiae was the dominant yeast host for heterologous protein production. Lately, other yeasts such as Komagataella sp., Kluyveromyces lactis, and Yarrowia lipolytica have emerged as advantageous hosts. In this review, a comparative analysis is done listing the advantages and disadvantages of using each host regarding the availability of genetic tools, strategies for cultivation in bioreactors, and the main techniques utilized for protein purification. Finally, examples of each host will be discussed regarding the total amount of protein recovered and its bioactivity due to correct folding and glycosylation patterns. Full article
(This article belongs to the Special Issue Recombinant Protein Expression in Microorganisms)
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