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Microbial Cell Factories May 2023The filamentous fungus Trichoderma reesei has been used as a host organism for the production of lignocellulosic biomass-degrading enzymes. Although this microorganism...
Inducer-free recombinant protein production in Trichoderma reesei: secretory production of endogenous enzymes and heterologous nanobodies using glucose as the sole carbon source.
BACKGROUND
The filamentous fungus Trichoderma reesei has been used as a host organism for the production of lignocellulosic biomass-degrading enzymes. Although this microorganism has high potential for protein production, it has not yet been widely used for heterologous recombinant protein production. Transcriptional induction of the cellulase genes is essential for high-level protein production in T. reesei; however, glucose represses this transcriptional induction. Therefore, cellulose is commonly used as a carbon source for providing its degraded sugars such as cellobiose, which act as inducers to activate the strong promoters of the major cellulase (cellobiohydrolase 1 and 2 (cbh1 and cbh2) genes. However, replacement of cbh1 and/or cbh2 with a gene encoding the protein of interest (POI) for high productivity and occupancy of recombinant proteins remarkably impairs the ability to release soluble inducers from cellulose, consequently reducing the production of POI. To overcome this challenge, we first used an inducer-free biomass-degrading enzyme expression system, previously developed to produce cellulases and hemicellulases using glucose as the sole carbon source, for recombinant protein production using T. reesei.
RESULTS
We chose endogenous secretory enzymes and heterologous camelid small antibodies (nanobody) as model proteins. By using the inducer-free strain as a parent, replacement of cbh1 with genes encoding two intrinsic enzymes (aspartic protease and glucoamylase) and three different nanobodies (1ZVH, caplacizumab, and ozoralizumab) resulted in their high secretory productions using glucose medium without inducers such as cellulose. Based on signal sequences (carrier polypeptides) and protease inhibitors, additional replacement of cbh2 with the nanobody gene increased the percentage of POI to about 20% of total secreted proteins in T. reesei. This allowed the production of caplacizumab, a bivalent nanobody, to be increased to 9.49-fold (508 mg/L) compared to the initial inducer-free strain.
CONCLUSIONS
In general, whereas the replacement of major cellulase genes leads to extreme decrease in the degradation capacity of cellulose, our inducer-free system enabled it and achieved high secretory production of POI with increased occupancy in glucose medium. This system would be a novel platform for heterologous recombinant protein production in T. reesei.
Topics: Cellulase; Glucose; Single-Domain Antibodies; Recombinant Proteins; Cellulose; Trichoderma
PubMed: 37208691
DOI: 10.1186/s12934-023-02109-y -
BioMed Research International 2022Enzymes play a powerful role as catalysts with high specificity and activity under mild environmental conditions. Significant hurdles, such as reduced solubility,... (Review)
Review
Enzymes play a powerful role as catalysts with high specificity and activity under mild environmental conditions. Significant hurdles, such as reduced solubility, reduced shelf-life, aggregate formation, and toxicity, are still ongoing struggles that scientists come across when purifying recombinant proteins. Over the past three decades, PEGylation techniques have been utilized to significantly overcome low solubility; increased protein stability, shelf-life, and bioactivity; and prevented protein aggregate formation. This review seeks to highlight the impact of PEG-based formulations that are significantly utilized to obtain favourable protein physiochemical properties. The authors further discuss other techniques that can be employed such as coexpression studies and nanotechnology-based skills to obtaining favourable protein physiochemical properties.
Topics: Drug Compounding; Polyethylene Glycols; Protein Stability; Recombinant Proteins; Solubility
PubMed: 35924267
DOI: 10.1155/2022/8929715 -
Microbial Cell Factories Sep 2021Escherichia coli is the most widely used bacterium in prokaryotic expression system for the production of recombinant proteins. In BL21 (DE3), the gene encoding the T7...
Escherichia coli is the most widely used bacterium in prokaryotic expression system for the production of recombinant proteins. In BL21 (DE3), the gene encoding the T7 RNA polymerase (T7 RNAP) is under control of the strong lacUV5 promoter (P), which is leakier and more active than wild-type lac promoter (P) under certain growth conditions. These characteristics are not advantageous for the production of those recombinant proteins with toxic or growth-burdened. On the one hand, leakage expression of T7 RNAP leads to rapid production of target proteins under non-inducing period, which sucks resources away from cellular growth. Moreover, in non-inducing or inducing period, high expression of T7 RNAP production leads to the high-production of hard-to-express proteins, which may all lead to loss of the expression plasmid or the occurrence of mutations in the expressed gene. Therefore, more BL21 (DE3)-derived variant strains with rigorous expression and different expression level of T7 RNAP should be developed. Hence, we replaced P with other inducible promoters respectively, including arabinose promoter (P), rhamnose promoter (P), tetracycline promoter (P), in order to optimize the production of recombinant protein by regulating the transcription level and the leakage level of T7 RNAP. Compared with BL21 (DE3), the constructed engineered strains had higher sensitivity to inducers, among which rhamnose and tetracycline promoters had the lowest leakage ability. In the production of glucose dehydrogenase (GDH), a protein that causes host autolysis, the engineered strain BL21 (DE3::ara) exhibited higher biomass, cell survival rate and foreign protein expression level than that of BL21 (DE3). In addition, these engineered strains had been successfully applied to improve the production of membrane proteins, including E. coli cytosine transporter protein (CodB), the E. coli membrane protein insertase/foldase (YidC), and the E. coli F-ATPase subunit b (Ecb). The engineered strains constructed in this paper provided more host choices for the production of recombinant proteins.
Topics: Cloning, Molecular; DNA-Directed RNA Polymerases; Escherichia coli; Gene Expression Regulation, Bacterial; Genetic Vectors; Membrane Transport Proteins; Protein Transport; Recombinant Proteins; Viral Proteins
PubMed: 34565359
DOI: 10.1186/s12934-021-01680-6 -
Advanced Healthcare Materials Jun 2019Proteins are versatile macromolecules that can perform a variety of functions. In the past three decades, they have been commonly used as building blocks to generate a... (Review)
Review
Proteins are versatile macromolecules that can perform a variety of functions. In the past three decades, they have been commonly used as building blocks to generate a range of biomaterials. Owing to their flexibility, proteins can either be used alone or in combination with other functional molecules. Advances in synthetic and chemical biology have enabled new protein fusions as well as the integration of new functional groups leading to biomaterials with emergent properties. This review discusses protein-engineered materials from the perspectives of domain-based designs as well as physical and chemical approaches for crosslinked materials, with special emphasis on the creation of hydrogels. Engineered proteins that organize or template metal ions, bear noncanonical amino acids (NCAAs), and their potential applications, are also reviewed.
Topics: Biocompatible Materials; Hydrogels; Protein Engineering; Recombinant Proteins
PubMed: 30938924
DOI: 10.1002/adhm.201801374 -
Macromolecular Bioscience Jul 2017Recombinant protein design allows modular protein domains with different functionalities and responsive behaviors to be easily combined. Inclusion of these protein... (Review)
Review
Recombinant protein design allows modular protein domains with different functionalities and responsive behaviors to be easily combined. Inclusion of these protein domains can enable recombinant proteins to have complex responses to their environment (e.g., temperature-triggered aggregation followed by enzyme-mediated cleavage for drug delivery or pH-triggered conformational change and self-assembly leading to structural stabilization by adjacent complementary residues). These "smart" behaviors can be tuned by amino acid identity and sequence, chemical modifications, and addition of other components. A wide variety of domains and peptides have smart behavior. This review focuses on protein designs for self-assembly or conformational changes due to stimuli such as shifts in temperature or pH.
Topics: Animals; Humans; Protein Aggregates; Protein Domains; Protein Engineering; Recombinant Proteins
PubMed: 28337848
DOI: 10.1002/mabi.201600554 -
FEBS Letters Jan 2014Expression of recombinant proteins in Escherichia coli (E. coli) remains the most popular and cost-effective method for producing proteins in basic research and for... (Review)
Review
Expression of recombinant proteins in Escherichia coli (E. coli) remains the most popular and cost-effective method for producing proteins in basic research and for pharmaceutical applications. Despite accumulating experience and methodologies developed over the years, production of recombinant proteins prone to aggregate in E. coli-based systems poses a major challenge in most research applications. The challenge of manufacturing these proteins for pharmaceutical applications is even greater. This review will discuss effective methods to reduce and even prevent the formation of aggregates in the course of recombinant protein production. We will focus on important steps along the production path, which include cloning, expression, purification, concentration, and storage.
Topics: Genetic Engineering; Protein Multimerization; Protein Structure, Quaternary; Recombinant Proteins
PubMed: 24211444
DOI: 10.1016/j.febslet.2013.10.044 -
Essays in Biochemistry Jul 2021Secreted recombinant proteins are of great significance for industry, healthcare and a sustainable bio-based economy. Consequently, there is an ever-increasing need for... (Review)
Review
Secreted recombinant proteins are of great significance for industry, healthcare and a sustainable bio-based economy. Consequently, there is an ever-increasing need for efficient production platforms to deliver such proteins in high amounts and high quality. Gram-positive bacteria, particularly bacilli such as Bacillus subtilis, are favored for the production of secreted industrial enzymes. Nevertheless, recombinant protein production in the B. subtilis cell factory can be very challenging due to bottlenecks in the general (Sec) secretion pathway as well as this bacterium's intrinsic capability to secrete a cocktail of highly potent proteases. This has placed another Gram-positive bacterium, Lactococcus lactis, in the focus of attention as an alternative, non-proteolytic, cell factory for secreted proteins. Here we review our current understanding of the secretion pathways exploited in B. subtilis and L. lactis to deliver proteins from their site of synthesis, the cytoplasm, into the fermentation broth. An advantage of this cell factory comparison is that it identifies opportunities for protein secretion pathway engineering to remove or bypass current production bottlenecks. Noteworthy new developments in cell factory engineering are the mini-Bacillus concept, highlighting potential advantages of massive genome minimization, and the application of thus far untapped 'non-classical' protein secretion routes. Altogether, it is foreseen that engineered lactococci will find future applications in the production of high-quality proteins at the relatively small pilot scale, while engineered bacilli will remain a favored choice for protein production in bulk.
Topics: Bacillus subtilis; Bacterial Proteins; Lactococcus lactis; Protein Engineering; Protein Transport; Recombinant Proteins
PubMed: 33955475
DOI: 10.1042/EBC20200171 -
Protein Science : a Publication of the... Sep 2023Recombinant human growth hormone (rhGH) and GH receptor antagonists (GHAs) are used clinically to treat a range of disorders associated with GH deficiency or... (Review)
Review
Recombinant human growth hormone (rhGH) and GH receptor antagonists (GHAs) are used clinically to treat a range of disorders associated with GH deficiency or hypersecretion, respectively. However, these biotherapeutics can be difficult and expensive to manufacture with multiple challenges from recombinant protein generation through to the development of long-acting formulations required to improve the circulating half-life of the drug. In this review, we summarize methodologies and approaches used for making and purifying recombinant GH and GHA proteins, and strategies to improve pharmacokinetic and pharmacodynamic properties, including PEGylation and fusion proteins. Therapeutics that are in clinical use or are currently under development are also discussed.
Topics: Humans; Human Growth Hormone; Recombinant Proteins; Receptors, Somatotropin
PubMed: 37428391
DOI: 10.1002/pro.4727 -
Microbial Cell Factories Apr 2022Proteins are used as reagents in a broad range of scientific fields. The reliability and reproducibility of experimental data will largely depend on the quality of the... (Review)
Review
BACKGROUND
Proteins are used as reagents in a broad range of scientific fields. The reliability and reproducibility of experimental data will largely depend on the quality of the (recombinant) proteins and, consequently, these should undergo thorough structural and functional controls. Depending on the downstream application and the biochemical characteristics of the protein, different sets of specific features will need to be checked.
RESULTS
A number of examples, representative of recurrent issues and previously published strategies, has been reported that illustrate real cases of recombinant protein production in which careful strategy design at the start of the project combined with quality controls throughout the production process was imperative to obtain high-quality samples compatible with the planned downstream applications. Some proteins possess intrinsic properties (e.g., prone to aggregation, rich in cysteines, or a high affinity for nucleic acids) that require certain precautions during the expression and purification process. For other proteins, the downstream application might demand specific conditions, such as for proteins intended for animal use that need to be endotoxin-free.
CONCLUSIONS
This review has been designed to act as a practical reference list for researchers who wish to produce and evaluate recombinant proteins with certain specific requirements or that need particular care for their preparation and storage.
Topics: Animals; Chromatography, Affinity; Recombinant Proteins; Reproducibility of Results
PubMed: 35392897
DOI: 10.1186/s12934-022-01778-5 -
Frontiers in Immunology 2018Recombinant proteins are gaining enormous importance these days due to their wide application as biopharmaceutical products and proven safety record. Various recombinant... (Review)
Review
Recombinant proteins are gaining enormous importance these days due to their wide application as biopharmaceutical products and proven safety record. Various recombinant proteins of therapeutic and prophylactic importance have been successfully produced in microbial and higher expression host systems. Since there is no specific antiviral therapy available against dengue, the prevention by vaccination is the mainstay in reducing the disease burden. Therefore, efficacious vaccines are needed to control the spread of dengue worldwide. Dengue is an emerging viral disease caused by any of dengue virus 1-4 serotypes that affects the human population around the globe. Dengue virus is a single stranded RNA virus encoding three structural proteins (capsid protein, pre-membrane protein, and envelope protein) and seven non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5). As the only licensed dengue vaccine (Dengvaxia) is unable to confer balanced protection against all the serotypes, therefore various approaches for development of dengue vaccines including tetravalent live attenuated, inactivated, plasmid DNA, virus-vectored, virus-like particles, and recombinant subunit vaccines are being explored. These candidates are at different stages of vaccine development and have their own merits and demerits. The promising subunit vaccines are mainly based on envelope or its domain and non-structural proteins of dengue virus. These proteins have been produced in different hosts and are being investigated for development of a successful dengue vaccine. Novel immunogens have been designed employing various strategies like protein engineering and fusion of antigen with various immunostimulatory motif to work as self-adjuvant. Moreover, recombinant proteins can be formulated with novel adjuvants to enhance the immunogenicity and thus conferring better protection to the vaccinees. With the advent of newer and safer host systems, these recombinant proteins can be produced in a cost effective manner at large scale for vaccine studies. In this review, we summarize recent developments in recombinant protein based dengue vaccines that could lead to a good number of efficacious vaccine candidates for future human use and ultimately alternative dengue vaccine candidates.
Topics: Dengue; Dengue Vaccines; Dengue Virus; Humans; Recombinant Proteins; Vaccines, Subunit; Vaccines, Synthetic; Viral Proteins
PubMed: 30190720
DOI: 10.3389/fimmu.2018.01919