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Heterologous Expression of Difficult to Produce Proteins in Bacterial Systems

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Microbiology".

Deadline for manuscript submissions: closed (30 November 2019) | Viewed by 50510

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Dr. Elena Garcia-Fruitos

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Department of Ruminant Production, Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Torre Marimon, 08140 Barcelona, Spain
Interests: antimicrobial alternatives; antimicrobial resistance; animal health; infectious disease
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Dr. Neus Ferrer-Miralles

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1. Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain
2. Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Barcelona, Spain
3. Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Barcelona, Spain
4. CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Barcelona, Spain
5. Plataforma de Producción de Proteínas, CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN) and Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Barcelona, Spain
Interests: recombinant protein; protein engineering; nanobiotechnology; microbiology; protein nanoparticles; protein aggregation; functional amyloids; cell-targeted delivery
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Dear Colleagues,

Many proteins of interest are produced in recombinant prokaryotic or eukaryotic expression systems. Among the prokaryotic expression systems, bacterial hosts are widely used for the production of recombinant proteins. Under overexpression conditions, the overproduced heterologous protein in recombinant bacteria can partition into two separate fractions: soluble and insoluble (also known as inclusion bodies). Often, the proteins of interest are difficult to express and, as a consequence, the final yields are unacceptable. In other cases, the proteins are prone to aggregation, making it challenging or impossible to obtain protein from the soluble fraction. In light of these issues, much of the research effort during the last few decades has gone towards the development of strategies to increase the efficiency of the production process for those difficult-to-obtain proteins. Thus, this Special Issue of IJMS will cover recent research activity towards the development of novel strategies used to obtain optimal yields of difficult-to-produce heterologous proteins which use bacterial expression systems as cell factories.

Dr. Elena Garcia-Fruitos
Dr. Neus Ferrer-Miralles
Guest Editors

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Keywords

recombinant proteins
heterologous proteins
difficult-to-produce proteins
bacterial expression systems
strategies for protein production
strategies for protein purification

Published Papers (10 papers)

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Editorial

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4 pages, 197 KiB

Open AccessEditorial

Heterologous Expression of Difficult to Produce Proteins in Bacterial Systems

by Neus Ferrer-Miralles and Elena Garcia-Fruitós

Int. J. Mol. Sci. 2024, 25(2), 822; https://doi.org/10.3390/ijms25020822 - 9 Jan 2024

Viewed by 862

Abstract

Proteins play a crucial role in maintaining homeostasis, providing structure, and enabling various functions in biological systems [...] Full article

(This article belongs to the Special Issue Heterologous Expression of Difficult to Produce Proteins in Bacterial Systems)

Research

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20 pages, 1034 KiB

Open AccessArticle

Scalable High-Performance Production of Recombinant Horseradish Peroxidase from E. coli Inclusion Bodies

by Diana Humer, Julian Ebner and Oliver Spadiut

Int. J. Mol. Sci. 2020, 21(13), 4625; https://doi.org/10.3390/ijms21134625 - 29 Jun 2020

Cited by 17 | Viewed by 4523

Abstract

Horseradish peroxidase (HRP), an enzyme omnipresent in biotechnology, is still produced from hairy root cultures, although this procedure is time-consuming and only gives low yields. In addition, the plant-derived enzyme preparation consists of a variable mixture of isoenzymes with high batch-to-batch variation preventing its use in therapeutic applications. In this study, we present a novel and scalable recombinant HRP production process in Escherichia coli that yields a highly pure, active and homogeneous single isoenzyme. We successfully developed a multi-step inclusion body process giving a final yield of 960 mg active HRP/L culture medium with a purity of ≥99% determined by size-exclusion high-performance liquid chromatography (SEC-HPLC). The Reinheitszahl, as well as the activity with 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 3,3′,5,5′-tetramethylbenzidine (TMB) as reducing substrates, are comparable to commercially available plant HRP. Thus, our preparation of recombinant, unglycosylated HRP from E. coli is a viable alternative to the enzyme from plant and highly interesting for therapeutic applications. Full article

(This article belongs to the Special Issue Heterologous Expression of Difficult to Produce Proteins in Bacterial Systems)

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14 pages, 2146 KiB

Open AccessArticle

Production of Active Recombinant Hyaluronidase Inclusion Bodies from Apis mellifera in E. coli Bl21(DE3) and characterization by FT-IR Spectroscopy

by Andreas Schwaighofer, Sarah Ablasser, Laurin Lux, Julian Kopp, Christoph Herwig, Oliver Spadiut, Bernhard Lendl and Christoph Slouka

Int. J. Mol. Sci. 2020, 21(11), 3881; https://doi.org/10.3390/ijms21113881 - 29 May 2020

Cited by 9 | Viewed by 4520

Abstract

The bacterium E. coli is one of the most important hosts for recombinant protein production. The benefits are high growth rates, inexpensive media, and high protein titers. However, complex proteins with high molecular weight and many disulfide bonds are expressed as inclusion bodies (IBs). In the last decade, the overall perception of these IBs being not functional proteins changed, as enzyme activity was found within IBs. Several applications for direct use of IBs are already reported in literature. While fluorescent proteins or protein tags are used for determination of IB activity to date, direct measurements of IB protein activity are scacre. The expression of recombinant hyaluronidase from Apis mellifera in E. coli BL21(DE3) was analyzed using a face centered design of experiment approach. Hyaluronidase is a hard to express protein and imposes a high metabolic burden to the host. Conditions giving a high specific IB titer were found at 25 °C at low specific substrate uptake rates and induction times of 2 to 4 h. The protein activity of hyaluronidase IBs was verified using (Fourier transform) FT-IR spectroscopy. Degradation of the substrate hyaluronan occurred at increased rates with higher IB concentrations. Active recombinant hyaluronidase IBs can be immediately used for direct degradation of hyaluronan without further down streaming steps. FT-IR spectroscopy was introduced as a method for tracking IB activity and showed differences in degradation behavior of hyaluronan dependent on the applied active IB concentration. Full article

(This article belongs to the Special Issue Heterologous Expression of Difficult to Produce Proteins in Bacterial Systems)

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14 pages, 1732 KiB

Open AccessArticle

Production of Extracellular Matrix Proteins in the Cytoplasm of E. coli: Making Giants in Tiny Factories

by Anil A. Sohail, Madhuri Gaikwad, Prakash Khadka, Mirva J. Saaranen and Lloyd W. Ruddock

Int. J. Mol. Sci. 2020, 21(3), 688; https://doi.org/10.3390/ijms21030688 - 21 Jan 2020

Cited by 13 | Viewed by 4887

Abstract

Escherichia coli is the most widely used protein production host in academia and a major host for industrial protein production. However, recombinant production of eukaryotic proteins in prokaryotes has challenges. One of these is post-translational modifications, including native disulfide bond formation. Proteins containing disulfide bonds have traditionally been made by targeting to the periplasm or by in vitro refolding of proteins made as inclusion bodies. More recently, systems for the production of disulfide-containing proteins in the cytoplasm have been introduced. However, it is unclear if these systems have the capacity for the production of disulfide-rich eukaryotic proteins. To address this question, we tested the capacity of one such system to produce domain constructs, containing up to 44 disulfide bonds, of the mammalian extracellular matrix proteins mucin 2, alpha tectorin, and perlecan. All were successfully produced with purified yields up to 6.5 mg/L. The proteins were further analyzed using a variety of biophysical techniques including circular dichroism spectrometry, thermal stability assay, and mass spectrometry. These analyses indicated that the purified proteins are most likely correctly folded to their native state. This greatly extends the use of E. coli for the production of eukaryotic proteins for structural and functional studies. Full article

(This article belongs to the Special Issue Heterologous Expression of Difficult to Produce Proteins in Bacterial Systems)

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21 pages, 4006 KiB

Open AccessArticle

Expression, Purification, and Characterization of Anti-Zika virus Envelope Protein: Polyclonal and Chicken-Derived Single Chain Variable Fragment Antibodies

by Pharaoh Fellow Mwale, Chi-Hsin Lee, Liang-Tzung Lin, Sy-Jye Leu, Yun-Ju Huang, Liao-Chun Chiang, Yan-Chiao Mao and Yi-Yuan Yang

Int. J. Mol. Sci. 2020, 21(2), 492; https://doi.org/10.3390/ijms21020492 - 13 Jan 2020

Cited by 14 | Viewed by 3263

Abstract

Zika virus (ZIKV) is a new and emerging virus that has caused outbreaks worldwide. The virus has been linked to congenital neurological malformations in neonates and Guillain–Barré syndrome in adults. Currently there are no effective vaccines available. As a result, there is a great need for ZIKV treatment. In this study, we developed single chain variable fragment (scFv) antibodies that target the ZIKV envelope protein using phage display technology. We first induced an immune response in white leghorn laying hens against the ZIKV envelope (E) protein. Chickens were immunized and polyclonal immunoglobulin yolk (IgY) antibodies were extracted from egg yolks. A high-level titer of anti-ZIKV_E IgY antibodies was detected using enzyme-linked immunosorbent assay (ELISA) after the third immunization. The titer persisted for at least 9 weeks. We constructed two antibody libraries that contained 5.3 × 10⁶ and 4.5 × 10⁶ transformants. After biopanning, an ELISA phage assay confirmed the enrichment of specific clones. We randomly selected 26 clones that expressed ZIKV scFv antibodies and classified them into two groups, short-linker and long-linker. Of these, four showed specific binding activities toward ZIKV_E proteins. These data suggest that the polyclonal and monoclonal scFv antibodies have the diagnostic or therapeutic potential for ZIKV. Full article

(This article belongs to the Special Issue Heterologous Expression of Difficult to Produce Proteins in Bacterial Systems)

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Review

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25 pages, 1052 KiB

Open AccessReview

Heterologous Hydrogenase Overproduction Systems for Biotechnology—An Overview

by Qin Fan, Peter Neubauer, Oliver Lenz and Matthias Gimpel

Int. J. Mol. Sci. 2020, 21(16), 5890; https://doi.org/10.3390/ijms21165890 - 16 Aug 2020

Cited by 15 | Viewed by 4862

Abstract

Hydrogenases are complex metalloenzymes, showing tremendous potential as H₂-converting redox catalysts for application in light-driven H₂ production, enzymatic fuel cells and H₂-driven cofactor regeneration. They catalyze the reversible oxidation of hydrogen into protons and electrons. The apo-enzymes are not active unless they are modified by a complicated post-translational maturation process that is responsible for the assembly and incorporation of the complex metal center. The catalytic center is usually easily inactivated by oxidation, and the separation and purification of the active protein is challenging. The understanding of the catalytic mechanisms progresses slowly, since the purification of the enzymes from their native hosts is often difficult, and in some case impossible. Over the past decades, only a limited number of studies report the homologous or heterologous production of high yields of hydrogenase. In this review, we emphasize recent discoveries that have greatly improved our understanding of microbial hydrogenases. We compare various heterologous hydrogenase production systems as well as in vitro hydrogenase maturation systems and discuss their perspectives for enhanced biohydrogen production. Additionally, activities of hydrogenases isolated from either recombinant organisms or in vivo/in vitro maturation approaches were systematically compared, and future perspectives for this research area are discussed. Full article

(This article belongs to the Special Issue Heterologous Expression of Difficult to Produce Proteins in Bacterial Systems)

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25 pages, 1521 KiB

Open AccessReview

Engineering Biology to Construct Microbial Chassis for the Production of Difficult-to-Express Proteins

by Kangsan Kim, Donghui Choe, Dae-Hee Lee and Byung-Kwan Cho

Int. J. Mol. Sci. 2020, 21(3), 990; https://doi.org/10.3390/ijms21030990 - 2 Feb 2020

Cited by 32 | Viewed by 7673

Abstract

A large proportion of the recombinant proteins manufactured today rely on microbe-based expression systems owing to their relatively simple and cost-effective production schemes. However, several issues in microbial protein expression, including formation of insoluble aggregates, low protein yield, and cell death are still highly recursive and tricky to optimize. These obstacles are usually rooted in the metabolic capacity of the expression host, limitation of cellular translational machineries, or genetic instability. To this end, several microbial strains having precisely designed genomes have been suggested as a way around the recurrent problems in recombinant protein expression. Already, a growing number of prokaryotic chassis strains have been genome-streamlined to attain superior cellular fitness, recombinant protein yield, and stability of the exogenous expression pathways. In this review, we outline challenges associated with heterologous protein expression, some examples of microbial chassis engineered for the production of recombinant proteins, and emerging tools to optimize the expression of heterologous proteins. In particular, we discuss the synthetic biology approaches to design and build and test genome-reduced microbial chassis that carry desirable characteristics for heterologous protein expression. Full article

(This article belongs to the Special Issue Heterologous Expression of Difficult to Produce Proteins in Bacterial Systems)

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21 pages, 2111 KiB

Open AccessReview

Escherichia coli Extract-Based Cell-Free Expression System as an Alternative for Difficult-to-Obtain Protein Biosynthesis

by Sviatlana Smolskaya, Yulia A. Logashina and Yaroslav A. Andreev

Int. J. Mol. Sci. 2020, 21(3), 928; https://doi.org/10.3390/ijms21030928 - 31 Jan 2020

Cited by 26 | Viewed by 9675

Abstract

Before utilization in biomedical diagnosis, therapeutic treatment, and biotechnology, the diverse variety of peptides and proteins must be preliminarily purified and thoroughly characterized. The recombinant DNA technology and heterologous protein expression have helped simplify the isolation of targeted polypeptides at high purity and their structure-function examinations. Recombinant protein expression in Escherichia coli, the most-established heterologous host organism, has been widely used to produce proteins of commercial and fundamental research interests. Nonetheless, many peptides/proteins are still difficult to express due to their ability to slow down cell growth or disrupt cellular metabolism. Besides, special modifications are often required for proper folding and activity of targeted proteins. The cell-free (CF) or in vitro recombinant protein synthesis system enables the production of such difficult-to-obtain molecules since it is possible to adjust reaction medium and there is no need to support cellular metabolism and viability. Here, we describe E. coli-based CF systems, the optimization steps done toward the development of highly productive and cost-effective CF methodology, and the modification of an in vitro approach required for difficult-to-obtain protein production. Full article

(This article belongs to the Special Issue Heterologous Expression of Difficult to Produce Proteins in Bacterial Systems)

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11 pages, 8100 KiB

Open AccessReview

Extremely Low Leakage Expression Systems Using Dual Transcriptional-Translational Control for Toxic Protein Production

by Yusuke Kato

Int. J. Mol. Sci. 2020, 21(3), 705; https://doi.org/10.3390/ijms21030705 - 21 Jan 2020

Cited by 19 | Viewed by 4074

Abstract

Expression systems for highly toxic protein genes must be conditional and suppress leakage expression to almost zero because even faint leakage expression may kill host cells, inhibit host growth, and cause loss of plasmids containing the toxic protein genes. The most widely used conditional expression systems are controlled only at the transcriptional level, and complete suppression of leakage expression is challenging. Recent progress on translational control has enabled construction of dual transcriptional-translational control systems in which leakage expression is strongly suppressed. This review summarizes the principles, features, and practical examples of dual transcriptional-translational control systems in bacteria, and provides future perspectives on these systems. Full article

(This article belongs to the Special Issue Heterologous Expression of Difficult to Produce Proteins in Bacterial Systems)

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14 pages, 2352 KiB

Open AccessReview

Heterologous and Homologous Expression of Proteins from Haloarchaea: Denitrification as Case of Study

by Rosa María Martínez-Espinosa

Int. J. Mol. Sci. 2020, 21(1), 82; https://doi.org/10.3390/ijms21010082 - 20 Dec 2019

Cited by 23 | Viewed by 4185

Abstract

Haloarchaea (halophilic microbes belonging to the Archaea domain) are microorganisms requiring mid or even high salt concentrations to be alive. The molecular machinery of these organisms is adapted to such conditions, which are stressful for most life forms. Among their molecular adaptations, halophilic proteins are characterized by their high content of acidic amino acids (Aspartate (Asp) and glumate (Glu)), being only stable in solutions containing high salt concentration (between 1 and 4 M total salt concentration). Recent knowledge about haloarchaeal peptides, proteins, and enzymes have revealed that many haloarchaeal species produce proteins of interest due to their potential applications in biotechnology-based industries. Although proteins of interest are usually overproduced in recombinant prokaryotic or eukaryotic expression systems, these procedures do not accurately work for halophilic proteins, mainly if such proteins contain metallocofactors in their structures. This work summarizes the main challenges of heterologous and homologous expression of enzymes from haloarchaea, paying special attention to the metalloenzymes involved in the pathway of denitrification (anaerobic reduction of nitrate to dinitrogen), a pathway with significant implications in wastewater treatment, climate change, and biosensor design. Full article

(This article belongs to the Special Issue Heterologous Expression of Difficult to Produce Proteins in Bacterial Systems)

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