scholarly journals Transformation of, and Heterologous Protein Expression in,Lactobacillus agilisandLactobacillus vaginalisIsolates from the Chicken Gastrointestinal Tract

2010 ◽  
Vol 77 (1) ◽  
pp. 220-228 ◽  
Author(s):  
David P. Stephenson ◽  
Robert J. Moore ◽  
Gwen E. Allison
Keyword(s):  
Gastrointestinal Tract ◽  
Protein Expression ◽  
Reporter Gene ◽  
Heterologous Protein ◽  
High Protein Diet ◽  
Plasmid Vector ◽  
Heterologous Protein Expression ◽  
Heterologous Proteins

ABSTRACTLactobacilli are naturally found in the gastrointestinal tract of chickens, and there is interest in utilizing autochthonous strains for the delivery of therapeutic proteins. Previously we identified three chicken-derivedLactobacillusstrains,Lactobacillus agilisLa3,Lactobacillus vaginalisLv5, andLactobacillus crispatusLc9, which persist in the gastrointestinal tract of chickens fed either a commercial or high-protein diet. In the current study, we investigated the ability to electrotransform these strains, determined plasmid vector stability, and compared reporter gene expression directed by several different promoters. The La3 and Lv5 strains were reproducibly transformed with efficiencies of 108and 106transformants per microgram of plasmid DNA, respectively. The third strain tested,L. crispatusLc9, was recalcitrant to all transformation protocols examined. The plasmid vectors pTRK563 and pTRKH2 were maintained over 100 generations in La3 and Lv5, respectively. The ability of La3 and Lv5 to express the heterologous reporter genegfpwas analyzed using heterologous and homologous promoters. Transformants of both La3 and Lv5 containing the La3ldhLpromoter were the most fluorescent. To our knowledge, this is the first report of successful transformation and heterologous protein expression inL. agilisandL. vaginalis. The ability of these strains to express heterologous proteinsin vitroindicates their potential utility asin vivodelivery vectors for therapeutic peptides to the chicken gastrointestinal tract.

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

2020 ◽  
Vol 21 (3) ◽  
pp. 990 ◽  
Author(s):  
Kangsan Kim ◽  
Donghui Choe ◽  
Dae-Hee Lee ◽  
Byung-Kwan Cho
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Proteins ◽  
Heterologous Protein ◽  
Recombinant Protein Expression ◽  
Cost Effective ◽  
Protein Yield ◽  
Heterologous Protein Expression ◽  
Heterologous Proteins ◽  
Expression Pathways

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.

scholarly journals Identification of Lactobacillus reuteri Genes Specifically Induced in the Mouse Gastrointestinal Tract

2003 ◽  
Vol 69 (4) ◽  
pp. 2044-2051 ◽  
Author(s):  
Jens Walter ◽  
Nicholas C. K. Heng ◽  
Walter P. Hammes ◽  
Diane M. Loach ◽  
Gerald W. Tannock ◽  
...  
Keyword(s):  
Gastrointestinal Tract ◽  
Reporter Gene ◽  
Stress Responses ◽  
Lactobacillus Reuteri ◽  
Xylose Isomerase ◽  
Methionine Sulfoxide ◽  
Methionine Sulfoxide Reductase ◽  
Chromosomal Dna

ABSTRACT Lactobacilli are common inhabitants of the gastrointestinal tracts of mammals and have received considerable attention due to their putative health-promoting properties. Little is known about the traits that enhance the ability of these bacteria to inhabit the gastrointestinal tract. In this paper we describe the development and application of a strategy based on in vivo expression technology (IVET) that enables detection of Lactobacillus reuteri genes specifically induced in the murine gut. A plasmid-based system was constructed containing ′ermGT (which confers lincomycin resistance) as the primary reporter gene for selection of promoters active in the gastrointestinal tract of mice treated with lincomycin. A second reporter gene, ′bglM (β-glucanase), allowed differentiation between constitutive and in vivo inducible promoters. The system was successfully tested in vitro and in vivo by using a constitutive promoter. Application of the IVET system with chromosomal DNA of L. reuteri 100-23 and reconstituted lactobacillus-free mice revealed three genes induced specifically during colonization. Two of the sequences showed homology to genes encoding xylose isomerase (xylA) and peptide methionine sulfoxide reductase (msrB), which are involved in nutrient acquisition and stress responses, respectively. The third locus showed homology to the gene encoding a protein whose function is not known. Our IVET system has the potential to identify genes of lactobacilli that have not previously been functionally characterized but which may be essential for growth of these bacteria in the gastrointestinal ecosystem.

Quantitative rt-pcr titration of recombinant semliki forest virus used for “in vitro” heterologous protein expression

2012 ◽  
Vol 29 ◽  
pp. S166-S167
Author(s):  
A.L. Puglia ◽  
A.G. Rezende ◽  
S.A.C. Jorge ◽  
R. Wagner ◽  
C.A. Pereira ◽  
...  
Keyword(s):  
Protein Expression ◽  
Heterologous Protein ◽  
Semliki Forest Virus ◽  
Heterologous Protein Expression ◽  
Rt Pcr

scholarly journals Overcoming codon-usage bias in heterologous protein expression in Streptococcus gordonii

Microbiology ◽  
2009 ◽  
Vol 155 (11) ◽  
pp. 3581-3588 ◽  
Author(s):  
Song F. Lee ◽  
Yi-Jing Li ◽  
Scott A. Halperin
Keyword(s):  
Protein Expression ◽  
Codon Usage ◽  
Codon Usage Bias ◽  
Heterologous Protein ◽  
Trna Gene ◽  
Heterologous Protein Expression ◽  
Streptococcus Gordonii ◽  
Heterologous Proteins ◽  
Single Chain ◽  
Rare Codons

One of the limitations facing the development of Streptococcus gordonii into a successful vaccine vector is the inability of this bacterium to express high levels of heterologous proteins. In the present study, we have identified 12 codons deemed as rare codons in S. gordonii and seven other streptococcal species. tRNA genes encoding 10 of the 12 rare codons were cloned into a plasmid. The plasmid was transformed into strains of S. gordonii expressing the fusion protein SpaP/S1, the anti-complement receptor 1 (CR1) single-chain variable fragment (scFv) antibody, or the Toxoplasma gondii cyclophilin C18 protein. These three heterologous proteins contained high percentages of amino acids encoded by rare codons. The results showed that the production of SpaP/S1, anti-CR1 scFv and C18 increased by 2.7-, 120- and 10-fold, respectively, over the control strains. In contrast, the production of the streptococcal SpaP protein without the pertussis toxin S1 fragment was not affected by tRNA gene supplementation, indicating that the increased production of SpaP/S1 protein was due to the ability to overcome the limitation caused by rare codons required for the S1 fragment. The increase in anti-CR1 scFv production was also observed in Streptococcus mutans following tRNA gene supplementation. Collectively, the findings in the present study demonstrate for the first time, to the best of our knowledge, that codon-usage bias exists in Streptococcus spp. and the limitation of heterologous protein expression caused by codon-usage bias can be overcome by tRNA supplementation.

scholarly journals A Novel Regulated Hybrid Promoter That Permits Autoinduction of Heterologous Protein Expression inKluyveromyces lactis

2019 ◽  
Vol 85 (14) ◽  
Author(s):  
Hassan Sakhtah ◽  
Juliane Behler ◽  
Alana Ali-Reynolds ◽  
Thomas B. Causey ◽  
Saulius Vainauskas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Carbon Source ◽  
Protein Expression ◽  
Recombinant Proteins ◽  
Heterologous Protein ◽  
Kluyveromyces Lactis ◽  
Heterologous Protein Expression ◽  
Heterologous Proteins ◽  
Content Type ◽  
Hybrid Promoter

ABSTRACTThe yeastKluyveromyces lactishas been a successful host for the production of heterologous proteins for over 30 years. Currently, the galactose-/lactose-inducible and glucose-repressibleLAC4promoter (PLAC4) is the most widely used promoter to drive recombinant protein expression inK. lactis. However, PLAC4is not fully repressed in the presence of glucose and significant protein expression still occurs. Thus, PLAC4is not suitable in processes where tight regulation of heterologous gene expression is required. In this study, we devised a novelK. lactispromoter system that is both strong and tightly controllable. We first tested several different endogenousK. lactispromoters for their ability to express recombinant proteins. A novel hybrid promoter (termed P350) was created by combining segments of twoK. lactispromoters, namely, the strong constitutive PGAP1promoter and the carbon source-sensitive PICL1promoter. We demonstrate that P350is tightly repressed in the presence of glucose or glycerol and becomes derepressed upon depletion of these compounds by the growing cells. We further illustrate the utility of P350-controlled protein expression in shake flask and high-cell-density bioreactor cultivation strategies. The P350hybrid promoter is a strong derepressible promoter for use in autoinduction of one-step fermentation processes for the production of heterologous proteins inK. lactis.IMPORTANCEThe yeastKluyveromyces lactisis an important host for the expression of recombinant proteins at both laboratory and industrial scales. However, the system lacks a tightly regulated promoter that permits controlled expression of heterologous proteins. In this study, we report the engineering of a highly regulated strong hybrid promoter (termed P350) for use inK. lactis. P350is tightly repressed by glucose or glycerol in the medium but strongly promotes gene expression once the carbon source has been consumed by the cells. This feature permits heterologous protein expression to be “autoinduced” at any scale without the addition of a gratuitous inducer molecule or changing feed solutions.

scholarly journals Upregulation of ETV2 Expression Promotes Endothelial Differentiation of Human Dental Pulp Stem Cells

2021 ◽  
Vol 30 ◽  
pp. 096368972097873
Author(s):  
Jing Li ◽  
Youming Zhu ◽  
Na Li ◽  
Tao Wu ◽  
Xianyu Zheng ◽  
...  
Keyword(s):  
Protein Expression ◽  
Dental Pulp ◽  
Tube Formation ◽  
Endothelial Differentiation ◽  
Cell Source ◽  
Lentiviral Infection ◽  
Mrna And Protein Expression ◽  
Human Dental Pulp

The lack of vasculogenesis often hampers the survivability and integration of newly engineered tissue grafts within the host. Autologous endothelial cells (ECs) are an ideal cell source for neovascularization, but they are limited by their scarcity, lack of proliferative capacity, and donor site morbidity upon isolation. The objective of this study was to determine whether differentiation of human dental pulp stem cells (DPSCs) into the endothelial lineage can be enhanced by recombinant ETV2 overexpression. DPSCs were extracted from fresh dental pulp tissues. ETV2 overexpression in DPSCs was achieved by lentiviral infection and cellular morphological changes were evaluated. The mRNA and protein expression levels of endothelial-specific markers were assessed through quantitative real-time polymerase chain reaction, western blot, immunofluorescence staining, and flow cytometry. The tube formation assay and Matrigel plug assay were also performed to evaluate the angiogenic potential of the ETV2-transduced cells in vitro and in vivo, respectively. Additionally, proteomic analysis was performed to analyze global changes in protein expression following ETV2 overexpression. After lentiviral infection, ETV2-overexpressing DPSCs showed endothelial-like morphology. Compared with control DPSCs, significantly higher mRNA and protein expression levels of endothelial-specific genes, including CD31, VE-Cadherin, VEGFR1, and VEGFR2, were detected in ETV2-overexpressing DPSCs. Moreover, ETV2 overexpression enhanced capillary-like tube formation on Matrigel in vitro, as well as neovascularization in vivo. In addition, comparative proteomic profiling showed that ETV2 overexpression upregulated the expression of vascular endothelial growth factor (VEGF) receptors, which was indicative of increased VEGF signaling. Taken together, our results indicate that ETV2 overexpression significantly enhanced the endothelial differentiation of DPSCs. Thus, this study shows that DPSCs can be a promising candidate cell source for tissue engineering applications.

scholarly journals Controlling Unconventional Secretion for Production of Heterologous Proteins in Ustilago maydis through Transcriptional Regulation and Chemical Inhibition of the Kinase Don3

2021 ◽  
Vol 7 (3) ◽  
pp. 179
Author(s):  
Kai P. Hussnaetter ◽  
Magnus Philipp ◽  
Kira Müntjes ◽  
Michael Feldbrügge ◽  
Kerstin Schipper
Keyword(s):  
Protein Production ◽  
Heterologous Protein ◽  
Ustilago Maydis ◽  
Downstream Processing ◽  
Culture Broth ◽  
Yeast Cells ◽  
Heterologous Protein Expression ◽  
Heterologous Proteins ◽  
Regulatory Systems ◽  
Unconventional Secretion

Heterologous protein production is a highly demanded biotechnological process. Secretion of the product to the culture broth is advantageous because it drastically reduces downstream processing costs. We exploit unconventional secretion for heterologous protein expression in the fungal model microorganism Ustilago maydis. Proteins of interest are fused to carrier chitinase Cts1 for export via the fragmentation zone of dividing yeast cells in a lock-type mechanism. The kinase Don3 is essential for functional assembly of the fragmentation zone and hence, for release of Cts1-fusion proteins. Here, we are first to develop regulatory systems for unconventional protein secretion using Don3 as a gatekeeper to control when export occurs. This enables uncoupling the accumulation of biomass and protein synthesis of a product of choice from its export. Regulation was successfully established at two different levels using transcriptional and post-translational induction strategies. As a proof-of-principle, we applied autoinduction based on transcriptional don3 regulation for the production and secretion of functional anti-Gfp nanobodies. The presented developments comprise tailored solutions for differentially prized products and thus constitute another important step towards a competitive protein production platform.

scholarly journals Honeysuckle Aqueous Extracts Induced let-7a Suppress EV71 Replication and Pathogenesis In Vitro and In Vivo and Is Predicted to Inhibit SARS-CoV-2

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 308
Author(s):  
Ying-Ray Lee ◽  
Chia-Ming Chang ◽  
Yuan-Chieh Yeh ◽  
Chi-Ying F. Huang ◽  
Feng-Mao Lin ◽  
...  
Keyword(s):  
Protein Expression ◽  
Western Blotting ◽  
Expression Profiles ◽  
Plaque Assay ◽  
Virus Titer ◽  
Enterovirus 71 ◽  
Luciferase Reporter ◽  
Pathogen Invasion

Honeysuckle (Lonicera japonica Thunb) is a traditional Chinese medicine (TCM) with an antipathogenic activity. MicroRNAs (miRNAs) are small non-coding RNA molecules that are ubiquitously expressed in cells. Endogenous miRNA may function as an innate response to block pathogen invasion. The miRNA expression profiles of both mice and humans after the ingestion of honeysuckle were obtained. Fifteen overexpressed miRNAs overlapped and were predicted to be capable of targeting three viruses: dengue virus (DENV), enterovirus 71 (EV71) and SARS-CoV-2. Among them, let-7a was examined to be capable of targeting the EV71 RNA genome by reporter assay and Western blotting. Moreover, honeysuckle-induced let-7a suppression of EV71 RNA and protein expression as well as viral replication were investigated both in vitro and in vivo. We demonstrated that let-7a targeted EV71 at the predicted sequences using luciferase reporter plasmids as well as two infectious replicons (pMP4-y-5 and pTOPO-4643). The suppression of EV71 replication and viral load was demonstrated in two cell lines by luciferase activity, RT-PCR, real-time PCR, Western blotting and plaque assay. Furthermore, EV71-infected suckling mice fed honeysuckle extract or inoculated with let-7a showed decreased clinical scores and a prolonged survival time accompanied with decreased viral RNA, protein expression and virus titer. The ingestion of honeysuckle attenuates EV71 replication and related pathogenesis partially through the upregulation of let-7a expression both in vitro and in vivo. Our previous report and the current findings imply that both honeysuckle and upregulated let-7a can execute a suppressive function against the replication of DENV and EV71. Taken together, this evidence indicates that honeysuckle can induce the expression of let-7a and that this miRNA as well as 11 other miRNAs have great potential to prevent and suppress EV71 replication.

scholarly journals 708 Application of a novel mSENS drug delivery technology for mRNA therapeutics

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A750-A750
Author(s):  
Sojin Lee ◽  
Joon Young Park ◽  
Goo-Young Kim ◽  
Sang Woo Jo ◽  
Minhyuk Yun ◽  
...  
Keyword(s):  
Particle Size ◽  
Protein Expression ◽  
Cancer Vaccine ◽  
Encapsulation Efficiency ◽  
Tumor Model ◽  
Cell Responses ◽  
Delivery Platform ◽  
Mrna Therapeutics

BackgroundSuccessful clinical translation of mRNA therapeutics requires an appropriate delivery strategy to overcome instability of mRNA and facilitate cellular uptake into the cells.1 Several lipid based nanoparticle approaches that encapsulate mRNA, notably lipid nanoparticle (LNP), have been developed, but their efficiency for delivery to certain target tissues and toxicity profiles still have room for improvement. The application of a novel polymer based nanoparticle technology platform, so called Stability Enhanced Nano Shells (SENS) for mRNA (mSENS) as a mRNA delivery platform for a cancer vaccine was demonstrated.MethodsThe physicochemical properties of mSENS formulation, particle size and encapsulation efficiency, were characterized using dynamic light scattering (DLS) and gel retardation assay. Using luciferase-encoding mRNA, the protein expression levels in vitro and in vivo were evaluated by luciferase assay or bioluminescence imaging (BLI), respectively. For cancer vaccine studies, antigen (tyrosinase-related protein 2 (Trp-2))-specific T cell responses were assessed by immunophenotyping mouse splenocytes using flow cytometry and by the enzyme-linked immunosorbent spot (ELISPOT) assay. The anti-tumor efficacy was studied in B16F10 lung tumor model in C57BL/6 mice. Liver and systemic toxicity of mSENS treated mice was evaluated through blood chemistry and complete blood count (CBC) tests.ResultsA library of mSENS formulations complexed with luciferase-encoding mRNA, were characterized for their particle size, surface charge, encapsulation efficiency, colloidal stability, and in vitro and in vivo luciferase protein expression level. Upon systemic administration in mice, varying biodistribution profiles were observed, implicating the potential for tailored delivery to target tissues. Particularly, cancer vaccine application was further developed leveraging the formulation with preferential spleen delivery. Following vaccination with Trp-2 mRNA encapsulated with mSENS (Trp-2 mRNA-mSENS) in B16F10 tumor bearing mice, strong Trp-2 antigen-specific IFN-γ T-cell responses were observed. Generated anti-tumor immunity also marked suppression of B16F10 lung tumors were observed in Trp-2-mSENS immunized mice compared to non-immunized controls, demonstrating the potential of mSENS as a mRNA delivery platform for the application for vaccine.ConclusionsProprietary biodegradable polymer based-mSENS platform offers an attractive delivery strategy for mRNA by tailoring to specific therapeutic applications. Depending on the application, whether it’s a vaccine or protein replacement, a rationally designed mSENS formulation can efficiently distribute mRNA to specific tissues. In particular, application of a splenic mSENS formulation for a cancer vaccine has been demonstrated in murine tumor model. In summary, mRNA delivery through mSENS platform is expected to provide significant opportunities in clinical development for mRNA therapeutics.Ethics ApprovalThe study was approved by Samyang Biopharmaceuticals’ IACUC (Institutional Animal Care and Use Committee), approval number SYAU-2027.ReferencePiotr S. Kowalski, Arnab Rudra, Lei Miao, and Daniel G. Anderson, delivering the messenger: advances in technologies for therapeutic mRNA delivery. Molecular Therapy Vol. 27 No 4 April 2019.

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