Established tools and emerging trends for the production of recombinant proteins and metabolites in Pichia pastoris

2021 ◽  
Author(s):  
Sonakshi De ◽  
Diethard Mattanovich ◽  
Pau Ferrer ◽  
Brigitte Gasser
Keyword(s):  
Pichia Pastoris ◽  
Recombinant Proteins ◽  
Protein Characterization ◽  
Pathway Engineering ◽  
Small Scale ◽  
Cell Factory ◽  
Scale Production ◽  
Heterologous Proteins ◽  
Metabolic Pathway Engineering

Abstract Besides bakers’ yeast, the methylotrophic yeast Komagataella phaffii (also known as Pichia pastoris) has been developed into the most popular yeast cell factory for the production of heterologous proteins. Strong promoters, stable genetic constructs and a growing collection of freely available strains, tools and protocols have boosted this development equally as thorough genetic and cell biological characterization. This review provides an overview of state-of-the-art tools and techniques for working with P. pastoris, as well as guidelines for the production of recombinant proteins with a focus on small-scale production for biochemical studies and protein characterization. The growing applications of P. pastoris for in vivo biotransformation and metabolic pathway engineering for the production of bulk and specialty chemicals are highlighted as well.

scholarly journals High-Yield Expression and Purification of Recombinant Influenza Virus Proteins from Stably-Transfected Mammalian Cell Lines

Vaccines ◽  
2020 ◽  
Vol 8 (3) ◽  
pp. 462
Author(s):  
Jeffrey W. Ecker ◽  
Greg A. Kirchenbaum ◽  
Spencer R. Pierce ◽  
Amanda L. Skarlupka ◽  
Rodrigo B. Abreu ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Cell Lines ◽  
Mammalian Cell ◽  
Recombinant Proteins ◽  
Influenza Viruses ◽  
Scale Production ◽  
Vaccine Platform ◽  
Mammalian Cell Lines

Influenza viruses infect millions of people each year, resulting in significant morbidity and mortality in the human population. Therefore, generation of a universal influenza virus vaccine is an urgent need and would greatly benefit public health. Recombinant protein technology is an established vaccine platform and has resulted in several commercially available vaccines. Herein, we describe the approach for developing stable transfected human cell lines for the expression of recombinant influenza virus hemagglutinin (HA) and recombinant influenza virus neuraminidase (NA) proteins for the purpose of in vitro and in vivo vaccine development. HA and NA are the main surface glycoproteins on influenza virions and the major antibody targets. The benefits for using recombinant proteins for in vitro and in vivo assays include the ease of use, high level of purity and the ability to scale-up production. This work provides guidelines on how to produce and purify recombinant proteins produced in mammalian cell lines through either transient transfection or generation of stable cell lines from plasmid creation through the isolation step via Immobilized Metal Affinity Chromatography (IMAC). Collectively, the establishment of this pipeline has facilitated large-scale production of recombinant HA and NA proteins to high purity and with consistent yields, including glycosylation patterns that are very similar to proteins produced in a human host.

scholarly journals NS1 Recombinant Proteins Are Efficiently Produced in Pichia pastoris and Have Great Potential for Use in Diagnostic Kits for Dengue Virus Infections

Diagnostics ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 379
Author(s):  
Mariana Fonseca Xisto ◽  
John Willians Oliveira Prates ◽  
Ingrid Marques Dias ◽  
Roberto Sousa Dias ◽  
Cynthia Canedo da Silva ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Dengue Virus ◽  
Recombinant Proteins ◽  
Large Scale ◽  
Limiting Factor ◽  
Scale Production ◽  
Global Public Health ◽  
Virus Infections ◽  
Correct Treatment ◽  
Diagnostic Kits

Dengue is one of the major diseases causing global public health concerns. Despite technological advances in vaccine production against all its serotypes, it is estimated that the dengue virus is responsible for approximately 390 million infections per year. Laboratory diagnosis has been the key point for the correct treatment and prevention of this disease. Currently, the limiting factor in the manufacture of dengue diagnostic kits is the large-scale production of the non-structural 1 (NS1) antigen used in the capture of the antibody present in the infected patients’ serum. In this work, we demonstrate the production of the non-structural 1 protein of dengue virus (DENV) serotypes 1–4 (NS1-DENV1, NS1-DENV2, NS1-DENV3, and NS1-DENV4) in the methylotrophic yeast Pichia pastoris KM71H. Secreted recombinant protein was purified by affinity chromatography and characterized by SDS-PAGE and ELISA. The objectives of this study were achieved, and the results showed that P. pastoris is a good heterologous host and worked well in the production of NS1DENV 1–4 recombinant proteins. Easy to grow and quick to obtain, this yeast secreted ready-to-use proteins, with a final yield estimated at 2.8–4.6 milligrams per liter of culture. We reached 85–91% sensitivity and 91–93% specificity using IgM as a target, and for anti-dengue IgG, 83–87% sensitivity and 81–93% specificity were achieved. In this work, we conclude that the NS1 recombinant proteins are efficiently produced in P. pastoris and have great potential for use in diagnostic kits for dengue virus infections. The transformed yeast obtained can be used for production in industrial-scale bioreactors.

scholarly journals Evolving small-molecule biosensors with improved performance and reprogrammed ligand specificity using OrthoRep

2021 ◽  
Author(s):  
Alex A Javanpour ◽  
Chang C Liu
Keyword(s):  
Small Molecule ◽  
High Throughput Screening ◽  
High Performance ◽  
Dynamic Range ◽  
Yeast Cells ◽  
Pathway Engineering ◽  
Muconic Acid ◽  
Metabolic Pathway Engineering ◽  
Operational Range

Genetically-encoded biosensors are valuable for the optimization of small-molecule biosynthesis pathways, because they transduce the production of small-molecule ligands into a readout compatible with high-throughput screening or selection in vivo. However, engineering biosensors with appropriate response functions and ligand specificities remains challenging. Here, we show that the continuous hypermutation system, OrthoRep, can be effectively applied to evolve biosensors with high dynamic range, reprogrammed activity towards desired non-cognate ligands, and proper operational range for coupling to biosynthetic pathways. In particular, we encoded the allosteric transcriptional factor, BenM, on OrthoRep such that propagation of host yeast cells resulted in BenM's rapid and continuous diversification. When these cells were subjected to cycles of culturing and sorting on BenM activity in the presence and absence of its cognate ligand, muconic acid, or the non-cognate ligand, adipic acid, we obtained multiple BenM variants that respond to their corresponding ligands. These biosensors outperform previously-engineered BenM-based biosensors by achieving substantially greater dynamic range (up to ~180-fold-induction) and broadened operational range. Expression of select BenM variants in the presence of a muconic acid biosynthetic pathway demonstrated sensitive biosensor activation without saturating response, which should enable pathway and host engineering for higher production of muconic and adipic acids. Given the streamlined manner in which high-performance and versatile biosensors were evolved using OrthoRep, this study provides a template for generating custom biosensors for metabolic pathway engineering and other biotechnology goals.

scholarly journals Fhl1p protein, a positive transcription factor in Pichia pastoris, enhances the expression of recombinant proteins

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Xueyun Zheng ◽  
Yimin Zhang ◽  
Xinying Zhang ◽  
Cheng Li ◽  
Xiaoxiao Liu ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Pichia Pastoris ◽  
Recombinant Proteins ◽  
Recombinant Protein Expression ◽  
Large Subunit ◽  
Protein A ◽  
Methylotrophic Yeast ◽  
Regulation Mechanism ◽  
Heterologous Proteins ◽  
Foreign Proteins

Abstract Background The methylotrophic yeast Pichia pastoris is well-known for the production of a broad spectrum of functional types of heterologous proteins including enzymes, antigens, engineered antibody fragments, and next gen protein scaffolds and many transcription factors are utilized to address the burden caused by the high expression of heterologous proteins. In this article, a novel P. pastoris transcription factor currently annotated as Fhl1p, an activator of ribosome biosynthesis processing, was investigated for promoting the expression of the recombinant proteins. Results The function of Fhl1p of P. pastoris for improving the expression of recombinant proteins was verified in strains expressing phytase, pectinase and mRFP, showing that the productivity was increased by 20–35%. RNA-Seq was used to study the Fhl1p regulation mechanism in detail, confirming Fhl1p involved in the regulation of rRNA processing genes, ribosomal small/large subunit biogenesis genes, Golgi vesicle transport genes, etc., which contributed to boosting the expression of foreign proteins. The overexpressed Fhl1p strain exhibited increases in the polysome and monosome levels, showing improved translation activities. Conclusion This study illustrated that the transcription factor Fhl1p could effectively enhance recombinant protein expression in P. pastoris. Furthermore, we provided the evidence that overexpressed Fhl1p was related to more active translation state.

scholarly journals Chemical Assistance in Refolding of Bacterial Inclusion Bodies

2011 ◽  
Vol 2011 ◽  
pp. 1-6 ◽  
Author(s):  
Mona Alibolandi ◽  
Hasan Mirzahoseini
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Inclusion Bodies ◽  
Industrial Processes ◽  
Small Scale ◽  
Heterologous Proteins ◽  
E Coli ◽  
Major Bottleneck ◽  
In Vitro Refolding

Escherichia coliis one of the most widely used hosts for the production of recombinant proteins but insoluble expression of heterologous proteins is a major bottleneck in production of recombinant proteins inE. coli.In vitrorefolding of inclusion body into proteins with native conformations is a solution for this problem but there is a need for optimization of condition for each protein specifically. Several approaches have been described for in vitro refolding; most of them involve the use of additives for assisting correct folding. Cosolutes play a major role in refolding process and can be classified according to their function as aggregation suppressors and folding enhancers. This paper presents a review of additives that are used in refolding process of insoluble recombinant proteins in small scale and industrial processes.

scholarly journals Genome-based engineering of ligninolytic enzymes in fungi

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Michael Dare Asemoloye ◽  
Mario Andrea Marchisio ◽  
Vijai Kumar Gupta ◽  
Lorenzo Pecoraro
Keyword(s):  
Synthetic Biology ◽  
Large Scale ◽  
Biomedical Application ◽  
Ligninolytic Enzymes ◽  
Fungal Species ◽  
Pathway Engineering ◽  
Main Body ◽  
Scale Production ◽  
Metabolic Pathway Engineering ◽  
Wide Range

Abstract Background Many fungi grow as saprobic organisms and obtain nutrients from a wide range of dead organic materials. Among saprobes, fungal species that grow on wood or in polluted environments have evolved prolific mechanisms for the production of degrading compounds, such as ligninolytic enzymes. These enzymes include arrays of intense redox-potential oxidoreductase, such as laccase, catalase, and peroxidases. The ability to produce ligninolytic enzymes makes a variety of fungal species suitable for application in many industries, including the production of biofuels and antibiotics, bioremediation, and biomedical application as biosensors. However, fungal ligninolytic enzymes are produced naturally in small quantities that may not meet the industrial or market demands. Over the last decade, combined synthetic biology and computational designs have yielded significant results in enhancing the synthesis of natural compounds in fungi. Main body of the abstract In this review, we gave insights into different protein engineering methods, including rational, semi-rational, and directed evolution approaches that have been employed to enhance the production of some important ligninolytic enzymes in fungi. We described the role of metabolic pathway engineering to optimize the synthesis of chemical compounds of interest in various fields. We highlighted synthetic biology novel techniques for biosynthetic gene cluster (BGC) activation in fungo and heterologous reconstruction of BGC in microbial cells. We also discussed in detail some recombinant ligninolytic enzymes that have been successfully enhanced and expressed in different heterologous hosts. Finally, we described recent advance in CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR associated) protein systems as the most promising biotechnology for large-scale production of ligninolytic enzymes. Short conclusion Aggregation, expression, and regulation of ligninolytic enzymes in fungi require very complex procedures with many interfering factors. Synthetic and computational biology strategies, as explained in this review, are powerful tools that can be combined to solve these puzzles. These integrated strategies can lead to the production of enzymes with special abilities, such as wide substrate specifications, thermo-stability, tolerance to long time storage, and stability in different substrate conditions, such as pH and nutrients.

scholarly journals Expression of Recombinant Factor IX Using the Transient Gene Expression Technique

2019 ◽  
Author(s):  
Jafar Vatandoost ◽  
Mohammad Amin Azimifar
Keyword(s):  
Gene Expression ◽  
Recombinant Proteins ◽  
Large Scale ◽  
Transient Gene Expression ◽  
Factor Ix ◽  
Cell Factory ◽  
Scale Production ◽  
Hek Cells ◽  
Large Scale Production ◽  
A Cell

Background: Pilot and large-scale production of recombinant proteins require the presence of stable clones, but the process of selecting stable clones is time consuming. Moreover, continuous clone culturing in large-scale production may cause loss of incoming plasmid and recombinant genes. Considering the advancements in Transient Gene Expression (TGE) technology, the large-scale expression of factor IX (FIX) was investigated in HEK cells by the TGE technique. Materials and Methods: HEK cells were seeded in a cell factory, and then transfected by pcDNA-hFIX plasmid using calcium phosphate co-precipitation method. Stable HEKhFIX cells were also seeded in a cell factory, separately. After adding vitamin K, recombinant FIX was quantified in conditioned media using an ELISA. Moreover, its functional activity was assayed using an aPTT test. Results: The results showed that the expression and activity of FIX by TGE technology was, respectively, 1.6 and 1.5 times higher than that obtained through stable HEK-FIX cells. Since calculating the specific activity revealed that for all time periods it is 0.2 mU/ng, so the increase in activity is due to the increase in the amount of FIX. Conclusions: HEK cells with higher transfectability seemed to be an appropriate alternative for transient expression for large-scale protein production. Furthermore, if rapid expression of recombinant proteins is intended, TGE can replace costly and lowyield methods.

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