Host Expression Systems Research Articles

The Development of Epitope-Based Recombinant Protein Vaccines against SARS-CoV-2.

The COVID-19 pandemic continues to cause infections and deaths, which are attributable to the SARS-CoV-2 Omicron variant of concern (VOC). Moderna's response to the declining protective efficacies of current SARS-CoV-2 vaccines against Omicron was to develop a bivalent booster vaccine based on the Spike (S) protein from the Wuhan and Omicron BA.4/BA.5 strains. This approach, while commendable, is unfeasible in light of rapidly emerging mutated viral strains. PubMed and Google Scholar were systematically reviewed for peer-reviewed papers up to January 2024. Articles included focused on specific themes such as the clinical history of recombinant protein vaccine development against different diseases, including COVID-19, the production of recombinant protein vaccines using different host expression systems, aspects to consider in recombinant protein vaccine development, and overcoming problems associated with large-scale recombinant protein vaccine production. In silico approaches to identify conserved and immunogenic epitopes could provide broad protection against SARS-CoV-2 VOCs but require validation in animal models. The recombinant protein vaccine development platform has shown a successful history in clinical development. Recombinant protein vaccines incorporating conserved epitopes may utilize a number of expression systems, such as yeast (Saccharomyces cerevisiae), baculovirus-insect cells (Sf9 cells), and Escherichia coli (E. coli). Current multi-epitope subunit vaccines against SARS-CoV-2 utilizing synthetic peptides are unfeasible for large-scale immunizations. Recombinant protein vaccines based on conserved and immunogenic proteins produced using E. coli offer high production yields, convenient purification, and cost-effective production of large-scale vaccine quantities capable of protecting against the SARS-CoV-2 D614G strain and its VOCs.

A Review: Development of a Synthetic Lactoferrin Biological System.

Lactoferrin is an iron-binding glycoprotein with antibacterial, antitumor, and immunomodulatory functions derived from milk and mucosal secretions. Lactoferrin is used in various products, such as infant formula milk powder, nutritional supplements, and cosmetics. Researchers have developed new technologies to produce lactoferrin because there are limitations in the separation and purification of lactoferrin from milk that cannot compensate for the market demand. Therefore, synthetic systems of lactoferrin have been developed with the development of genetic engineering, and the structure of lactoferrin expressed in heterologous systems is very similar to that of natural lactoferrin. The structure and functions of lactoferrin and the design and construction of synthetic lactoferrin biological systems, especially microbial synthetic systems, including prokaryotic and eukaryotic host-expression systems, are described. On the basis of these results, we summarize the challenges and solutions for constructing systems of high-yield lactoferrin. The development directions of recombinant lactoferrin are discussed in this review. Overall, the design and development of these synthetic biological systems have allowed us to explore the great potential of the industrial large-scale preparation of lactoferrin.

Alternative secretory signal sequences for recombinant protein production in Pichia pastoris

Extracellular protein production is primarily preferred to facilitate the downstream processes in recombinant protein production. Secretion of recombinant proteins is mediated by the processing of signal peptides in their N-terminal portion by the secretory mechanism of host expression systems. These molecular elements involved in secretion are functionally interchangeable between different species and secretion sequence screening is one of the widely used approaches to improve extracellular protein production. In this study, α-mating and protein internal repeats (PIR) secretion sequences isolated from different yeasts (Kluyveromyces lactis, Kluyveromyces marxianus and Hansenula polymorpha) were tested in Pichia pastoris for the production of two different proteins (α-amylase and xylanase) and compared with the well-known secretory signals, S. cerevisiae α-mating factor (Sc-MF) and P. pastoris protein internal repeats PIR (PpPIR). The results obtained showed the potential of signal sequences tested. Among the tested peptides, the highest yields were achieved with H. polymorpha protein internal repeats (HpPIR) and K. lactis α-mating factor (Kl-MF) for xylanase and K. marxianus protein internal repeats (KmPIR) and K. lactis α-mating factor (Kl-MF) for amylase. In further studies, these sequences can be evaluated as alternatives in the production of different proteins in P. pastoris and in the production of recombinant proteins in different expression systems.

Cost-effective Purification of Membrane Proteins using a Dual-detergent Strategy.

Understanding the mechanisms of membrane protein function is critical for biomedical research and drug discovery as membrane proteins constitute ∼30% of the proteins encoded by the genomes of both lower and higher organisms and are targets for two-thirds of approved drugs worldwide. Significant progress has been made in engineering host expression systems for large-scale production of membrane proteins and in determining their three-dimensional high-resolution structures. Despite these efforts, the study of membrane proteins at the atomic level is challenging due to poor expression and extraction, low yields of functional protein, and the complexity and heterogeneity of source membranes. Structural and spectroscopic studies of any membrane protein require that the protein be extracted from its native membranes into a membrane-mimetic stable environment, which is often achieved by the use of detergents. Unfortunately, there is no magic detergent that can extract all membrane proteins and successful extraction often requires a thorough screen of detergents. Furthermore, membrane protein purification in general and the detergents used are very expensive, which puts a financial constraint on sophisticated membrane protein studies. To overcome this hurdle, a dual-detergent strategy has recently been developed and successfully applied to purify various classes of pure, stable, and functionally relevant membrane proteins in a cost-effective manner. This strategy uses an inexpensive detergent for solubilization of the desired protein from membranes and a second detergent during protein purification. In the Basic Protocol, we describe the dual-detergent strategy to significantly reduce the overall purification cost of a bacterial membrane protein using the magnesium ion channel MgtE as an example. Support Protocols are also provided for selecting a suitable E. coli strain for protein expression and the optimal detergent(s) for membrane protein solubilization. © 2022 Wiley Periodicals LLC. Basic Protocol: Expression, membrane solubilization, and cost-effective purification of MgtE Support Protocol 1: Selecting a suitable E. coli strain for optimal protein expression Support Protocol 2: Identification of suitable detergents for membrane protein solubilization.

Engineering a carbohydrate-binding module to increase the expression level of glucoamylase in Pichia pastoris

BackgroundGlucoamylase is an important industrial enzyme for the saccharification of starch during sugar production, but the production cost of glucoamylase is a major limiting factor for the growth of the starch-based sugar market. Therefore, seeking strategies for high-level expression of glucoamylase in heterologous hosts are considered as the main way to reduce the enzyme cost.ResultsReGa15A from Rasamsonia emersonii and TlGa15B-GA2 from Talaromyces leycettanus have similar properties. However, the secretion level of ReGa15A was significantly higher than TlGa15B-GA2 in Pichia pastoris. To explore the underlying mechanisms affecting the differential expression levels of glucoamylase in P. pastoris, the amino acid sequences and three-dimensional structures of them were compared and analyzed. First, the CBM region was identified by fragment replacement as the key region affecting the expression levels of ReGa15A and TlGa15B-GA2. Then, through the substitution and site-directed mutation of the motifs in the CBM region, three mutants with significantly increased expression levels were obtained. The eight-point mutant TlGA-M4 (S589D/Q599A/G600Y/V603Q/T607I/V608L/N609D/R613Q), the three-point mutant TlGA-M6 (Q599A/G600Y/V603Q) and the five-point mutant TlGA-M7 (S589D/T607I/V608L/N609D/R613Q) have the same specific activity with the wild-type, and the enzyme activity and secretion level have increased by 4–5 times, respectively. At the same time, the expression levels were 5.8-, 2.0- and 2.4-fold higher than that of wild type, respectively. Meanwhile, the expression of genes related to the unfolded protein responses (UPR) in the endoplasmic reticulum (ER) did not differ significantly between the mutants and wild type. In addition, the most highly expressed mutant, TlGA-M7 exhibits rapidly and effectively hydrolyze raw corn starch.ConclusionsOur results constitute the first demonstration of improved expression and secretion of a glucoamylase in P. pastoris by introducing mutations within the non-catalytic CBM. This provides a novel and effective strategy for improving the expression of recombinant proteins in heterologous host expression systems.

Protein Expression Optimization Strategies in E. coli: A Tailored Approach in Strain Selection and Parallelizing Expression Conditions.

Escherichia coli remains a traditional and widely used host organism for recombinant protein production. Its well-studied genome, availability of vectors and strains, cheap and relatively straight-forward cultivation methods paired with reported high protein yields are reasons why E. coli is often the first-choice host expression system for recombinant protein production. The chapter enclosed here details protocols and design strategies in strain selection and methods on how to parallelize expression conditions to optimize for soluble target protein expression in E. coli. The methods described have been validated in a protein production research facility.

Heterologous Expression of Interferon α-2b in Lactococcus lactis and its Biological Activity against Colorectal Cancer Cells

Lita Meilina, Sri Budiarti , Apon Zaenal Mustopa*, Huda Shalahudin Darusman, Lita Triratna, Muhammad Ajietuta Nugraha, Muhammad Sabiq Bilhaq, and Ratih Asmana Ningrum. Microbiol. Biotechnol. Lett. 2021;49:75-87. https://doi.org/10.48022/mbl.2009.09008

Functional evaluation of a recombinant fungal immunomodulatory protein from L. rhinocerus produced in P. pastoris and E. coli host expression systems

Fungal immunomodulatory proteins (FIPs) are bioactive proteins with immunomodulatory properties. We previously reported the heterologous production in Escherichia coli of FIP-Lrh from Tiger milk mushroom (Lignosus rhinocerus) with potent cytotoxic effect on cancer cell lines. However, protein produced in E. coli lacks post-translational modifications and may be contaminated with lipopolysaccharide (LPS) endotoxin. Therefore, in this study, yFIP-Lrh produced in Pichia pastoris was functionally compared with eFIP-Lrh produced in E. coli. Expression construct of FIP-Lrh cDNA in pPICZα was generated, transformed into P. pastoris X-33 and Mut+ transformants were verified by colony PCR. Induction with 0.5% or 1% methanol resulted in a secreted 13.6 kDa yFIP-Lrh which was subsequently purified and verified using LCMS/MS analysis. Size exclusion chromatography confirmed eFIP-Lrh as a homodimer whereas the larger size of yFIP-Lrh may indicate post-translational modification despite negative for glycoproteins staining. At lower concentration (4-8 μg/mL), yFIP-Lrh induced significantly higher Th1 (IFN-γ, TNF-α) and Th2 (IL-6, IL-4, IL-5, IL-13) cytokines production in mice splenocytes, whereas 16 μg/mL eFIP-Lrh induced significantly higher pro-inflammatory cytokines (TNF-α, IL-6, IL-10), possibly due to higher residual LPS endotoxin (0.082 EU/mL) in eFIP-Lrh compared to negligible level in yFIP-Lrh (0.001 EU/mL). Furthermore, yFIP-Lrh showed higher cytotoxic effect on MCF-7 and HeLa cancer cells. Since both recombinant proteins of FIP-Lrh have the same peptide sequence, besides glycosylation, other post-translational modifications in yFIP-Lrh may account for its enhanced immunomodulatory and anti-proliferative activities. In conclusion, P. pastoris is preferred over E. coli for production of a functionally active yFIP-Lrh devoid of endotoxin contamination. KEY POINTS: • FIP-Lrh can induced production of Th1 and Th2 cytokines by mouse splenocytes. • Higher cytotoxic effect on cancer cells observed for yeast compared to E. coli produced FIP-Lrh. • P. pastoris allows production of an endotoxin-free and functionally active recombinant FIP-Lrh.

New Set of Yeast Vectors for Shuttle Expression in Escherichia coli

Promoters that play an essential role in the gene regulation are of particular interest to the synthetic biology communities. Recent advances in high-throughput DNA sequencing have greatly increased the breadth of new genetic parts. The development of promoters with broad host properties could enable rapid phenotyping of genetic constructs in different hosts. In this study, we discovered that the GAL1/10 bidirectional promoter from Saccharomyces cerevisiae could be used for shuttle expression in Escherichia coli. Further investigation revealed that the GAL1/10 bidirectional promoter is subjected to catabolite repression in E. coli. We next constructed a set of Golden-Gate assembly vectors for shuttle expression between S. cerevisiae and E. coli. The utility of shuttle vectors was demonstrated for rapid phenotyping of a multigene pathway for cinnamyl alcohol production. Taken together, our work opens a new avenue for the future development of broad host expression systems between prokaryotic and eukaryotic hosts.

Nicotiana benthamiana as a Transient Expression Host to Produce Auxin Analogs.

Plant secondary metabolites have applications for the food, biofuel, and pharmaceutical industries. Recent advances in pathway elucidation and host expression systems now allow metabolic engineering of plant metabolic pathways to produce “new-to-nature” derivatives with novel biological activities, thereby amplifying the range of industrial uses for plant metabolites. Here we use a transient expression system in the model plant Nicotiana benthamiana to reconstitute the two-step plant-derived biosynthetic pathway for auxin (indole acetic acid) to achieve accumulation up to 500 ng/g fresh mass (FM). By expressing these plant-derived enzymes in combination with either bacterial halogenases and alternative substrates, we can produce both natural and new-to-nature halogenated auxin derivatives up to 990 ng/g FM. Proteins from the auxin synthesis pathway, tryptophan aminotransferases (TARs) and flavin-dependent monooxygenases (YUCs), could be transiently expressed in combination with four separate bacterial halogenases to generate halogenated auxin derivatives. Brominated auxin derivatives could also be observed after infiltration of the transfected N. benthamiana with potassium bromide and the halogenases. Finally, the production of additional auxin derivatives could also be achieved by co-infiltration of TAR and YUC genes with various tryptophan analogs. Given the emerging importance of transient expression in N. benthamiana for industrial scale protein and product expression, this work provides insight into the capacity of N. benthamiana to interface bacterial genes and synthetic substrates to produce novel halogenated metabolites.

COVID-19: Studying the Global Pandemic – Foreword

COVID-19: Studying the Global Pandemic – Foreword

Regulatory outlook from concept to commercialization of Biosimilars in Brazil market

Biological products or biopharmaceuticals are medicinal products derived from living organism systems and manufactured by using modern biotechnology that differ widely from the conventional synthetic drugs. They are much larger and more complex molecules with inherent diversity; hence, different manufacturers cannot produce identical biological products, even with the same type of host expression system and equivalent technologies. Thus, biologics manufactured and marketed after patent expiration are usually referred to as biosimilars. Biosimilars endeavor to copy the original technology leading to the production of innovative biotechnological medicines to obtain a product which is similar to the reference product. These products reported to improve the treatment landscape for multiple diseases, particularly in the areas of oncology, blood disorders, rheumatology, endocrinology and are becoming choice of treatment regimen due to policy push by governments for it’s affordability without comprising on quality, safety and efficacy. Pharmaceutical exports from Brazil increased by around 41% between 2009 and 2013, touching a high of U.S. $1.516 billion. The valuation of Brazilian pharma markets has shown double digit growth in the past decade. Between 2012 and 2015, market valuations have increased from U.S. $25.2 billion to U.S. $35.3 billion.
 Biosimilarity is based on a comprehensive comparability exercise wherein unavoidable clinical differences are evaluated and must meet equivalence or non-inferiority criteria. Biosimilars need to comply with different regulatory requirements for market authorization in different sites. There are several other related issues that need to be defined by the national authorities, such as interchangeability, labeling and prescribing information. The Brazilian health surveillance agency shadows the key principles established by the World Health Organization for the assessment of biosimilarity. However, the regulations also widen the gap by having standalone application pathway that does not require the usual comparability exercise with the reference product, originating non-biosimilar copies. Interchangeability and the use of nonproprietary names are not regulated. The objective of this manuscript is to explore the Brazilian Regulatory outlook from concept to commercialization of Biosimilars.

Host expression system modulates recombinant Hsp70 activity through post-translational modifications.

The use of model organisms for recombinant protein production results in the addition of model-specific post-translational modifications (PTMs) that can affect the structure, charge, and function of the protein. The 70-kDa heat shock proteins (Hsp70) were originally described as intracellular chaperones, with ATPase and foldase activity. More recently, new extracellular activities of Hsp70 proteins (e.g. as immunomodulators) have been identified. While some studies indicate an inflammatory potential for extracellular Hsp70 proteins, others suggest an immunosuppressive activity. We hypothesized that the production of recombinant Hsp70 in different expression systems would result in the addition of different PTMs, perhaps explaining at least some of these opposing immunological outcomes. We produced and purified Mycobacterium tuberculosis DnaK from two different systems, Escherichia coli and Pichia pastoris, and analyzed by mass spectrometry the protein preparations, investigating the impact of PTMs in an in silico and in vitro perspective. The comparisons of DnaK structures in silico highlighted that electrostatic and topographical differences exist that are dependent upon the expression system. Production of DnaK in the eukaryotic system dramatically affected its ATPase activity, and significantly altered its ability to downregulate MHC II and CD86 expression on murine dendritic cells (DCs). Phosphatase treatment of DnaK indicated that some of these differences related specifically to phosphorylation. Altogether, our data indicate that PTMs are an important characteristic of the expression system, with differences that impact interactions of Hsps with their ligands and subsequent functional activities.

The fit of codon usage of human-isolated avian influenza A viruses to human

Avian influenza A viruses (AIVs) classify into 18 hemagglutinin (HA) and 11 neuraminidase (NA) subtypes. Even though H1N1 and H3N2 subtypes usually circulate among humans leading to infection, occasionally, H5, H6, H7, H9, and H10 that circulate in poultry also infect humans, and especially H5N1 and H7N9. Efficient virus replication is a critical factor that influences infection. Codon usage of a virus must coevolve with its host for efficient viral replication, therefore, we conduct a comprehensive analysis of codon usage bias in human-isolated AIVs to test their adaptation to host expression system. The relative synonymous codon usage (RSCU) pattern, and the codon adaptation index (CAI) are calculated for this purpose. We find that all human-isolated AIVs tend to eliminate GC and CpG compositions, which may prevent activation of the host innate immune system. Although codon usage differs between AIV subtypes, our data support the conclusion that natural selection has played a major role and mutation pressure a minor role in shaping codon usage bias in all AIVs. Our efforts discover that codon usage of genes encoding surface proteins of H5N1, and the polymerase genes of H7N9 has better fit to the human expression system. This may associate with their better replication and infection in human.

Membrane Protein Production and Purification from Escherichia coli and Sf9 Insect Cells.

A major obstacle to studying membrane proteins by biophysical techniques is the difficulty in producing sufficient amounts of materials for functional and structural studies. To overexpress the target membrane protein heterologously, especially an eukaryotic protein, a key step is to find the optimal host expression system and perform subsequent expression optimization. In this chapter, we describe protocols for screening membrane protein production using bacterial and insect cells, solubilization screening, large-scale production, and commonly used affinity chromatography purification methods. We discuss general optimization conditions, such as promoters and tags, and describe current techniques that can be used in any laboratory without specialized expensive equipment. Especially for insect cells, GFP fusions are particularly useful for localization and in-gel fluorescence detection of the proteins on SDS-PAGE. We give detailed protocols that can be used to screen the best expression and purification conditions for membrane protein study.

Strengths and weaknesses of the Brazilian regulation on biosimilars: A critical view of the regulatory requirements for biosimilars in Brazil.

Biological products or biopharmaceuticals are medicinal products derived from living systems and manufactured by modern biotechnological methods that differ widely from the traditional synthetic drugs. Monoclonal antibodies are the most rapidly growing type of biologic. They are much larger and more complex molecules with inherent diversity; therefore, different manufacturers cannot produce identical biological products, even with the same type of host expression system and equivalent technologies. Thus, legal follow-on biologics manufactured and marketed after patent expiration are usually referred to as biosimilars. Biosimilarity is based on a comparability exercise whereby unavoidable clinical differences are evaluated and must meet equivalence or non-inferiority criteria. Biosimilars need to comply with different regulatory requirements for market authorization in different sites. There are several other related issues that need to be defined by the national authorities, such as interchangeability, labeling and prescribing information. The Brazilian health surveillance agency follows the key principles established by the World Health Organization for the assessment of biosimilarity, although does not adopt the name ‘biosimilar’. However, the agency also made a compromise on a standalone application pathway that does not require the usual comparability exercise with the reference product, originating nonbiosimilar copies. Interchangeability and the use of nonproprietary names are not regulated, giving rise to pressures on physicians and conflicts of interest in the decision making on biosimilar use. The scope of this article is to present the Brazilian regulation on biosimilars, its strengths and weaknesses, and to discuss it in the face of regulations in the USA and Europe.

Codon Usage Bias of the Wheat Flower Development Gene WAG-2 and Other AGAMOUS Group Genes

Analyzing codon usage bias of WAG-2 gene in wheat three-pistil (TP) mutant may provide a basis for selecting the appropriate host expression systems to improve the expression of target genes. In the present study, we analyzed the codon bias of the complete coding sequence (CDS) of the WAG-2 gene in TP using Codon W program, and compared the results with AGAMOUS (AG) group genes of other plant species. Results showed that the WAG-2 gene in TP and other monocot AG group genes preferably used codons ending with G/C bases, but Arabidopsis thaliana, Nicotiana tabacum, and other dicot crops were biased toward the synonymous codons with A/T. The clustering results based on codon bias were consistent with those based on CDS of the AG group genes, indicating that the difference in codon preference of AG group genes sequences was closely associated with the genetic relationship of the species. The Euclidean distance coefficients of WAG-2 with A. thaliana and N. tabacum were 9.255 and 5.730, respectively, indicating that N. tabacum may be more suitable for the expression of WAG-2. There were 37 codons showing distinct usage differences between WAG-2 and genome of yeast, 23 between WAG-2 and Escherichia coli. Therefore, the E. coli was the superior protein expression system. These results may improve our understanding of codon usage bias and functional studies of WAG-2.

RETRACTED: Expression system for heterologous protein expression in the filamentous fungus Aspergillus unguis

Heterologous protein expression in filamentous fungi is advantageous, especially in the context of large scale production of high volume low value recombinant proteins. However, such systems are rare and not available in public domain. A novel filamentous fungus – Aspergillus unguis NII 08123 was used as host for developing a protein expression system. An expression cassette was assembled using A. nidulans glyceraldehyde 3 phosphate dehydrogenase promoter (Pgapd), tryptophan synthase transcription terminator (TtrpC) and hygromycin resistance gene (hph) as selection marker. The enhanced green fluorescent protein (GFP) gene from Aequorea victoria was used as the model test protein for the evaluation of the expression system. The genetic transformation of this novel fungus was optimized through electroporation. Use of heterologous signal peptides resulted in high levels of secreted expression. The fungal host-expression system combination was tested successfully for the expression of the recombinant therapeutic protein-human interferon beta (HuIFNβ).

Enhanced translation initiation factor 4G levels correlate with production levels of monoclonal antibodies in recombinant CHO cell lines.

Using cells to manufacture protein-based therapeutics or biopharmaceuticals is a rapidly expanding industrial activity. Chinese hamster ovary (CHO) cells are the most frequently used mammalian host-expression system for the manufacture of biopharmaceuticals. Over the past ∼30years academic and industrial researchers have studied cell expression characteristics with aims to improve product yield, quality, scalability and reproducibility. Although many steps in the gene expression and secretion pathways have been optimized, little attention has been paid to optimizing protein synthesis factors and regulators during this process. A new study in Biochemical Journal by Mead et al., provides a first systematic study of several protein synthesis factors and finds that the expression level of eIF4G1 correlates with the level of recombinant protein expressed in cultures. Optimizing levels and activities of protein synthesis factors may help to enhance recombinant protein expression of biopharmaceuticals.

Uncovering Plant Growth-Mediating Allelochemicals Produced by Soil Microorganisms

Coevolving interactions between a plant population and its microbiota can potentially yield a rhizosphere enriched in metagenomes containing the blueprints for a vast array of natural products. We describe a method of isolating those metabolites through activity-based screening of soil metagenomic libraries. The method allows for the isolation of small molecules produced in vector–host expression systems containing large-insert DNA fragments extracted from the target plant rhizospheres. Allelopathic activities derived from selected clones were screened against a series of controls. Nonmetric multidimensional scaling (NMS) showed similar effects of the set of controls on lettuce growth, whereas annual bluegrass had a broader range of growth responses. Methanol extracts from clones indicating activity showed distinct patterns in grass seedling growth from the empty vector control, but the same extracts showed no effect on lettuce. The results indicate that the metagenomics method and bioassay screen of clone extracts are tools that can be used for initial determination of allelopathic activity from noncultured soil microbiota.