scholarly journals Stable recombinant gene expression from a Ligilactobacillus live bacterial vector via chromosomal integration

2021 ◽  
Author(s):  
Ben Vezina ◽  
Theo Allnutt ◽  
Anthony L. Keyburn ◽  
Ben Wade ◽  
Thi Thu Hao Van ◽  
...  
Keyword(s):  
Gastrointestinal Tract ◽  
Protein Expression ◽  
Clostridium Perfringens ◽  
Recombinant Proteins ◽  
Therapeutic Proteins ◽  
Alternative Methods ◽  
Future Application ◽  
Expression Systems ◽  
Recombinant Gene Expression ◽  
Vaccine Antigens

Disease control in animal production systems requires constant vigilance. Historically, the application of in-feed antibiotics to control bacteria and improve performance has been a much-used approach to maintain animal health and welfare. However, the widespread use of in-feed antibiotics is thought to increase the risk of antibiotic resistance developing. Alternative methods to control disease and maintain productivity need to be developed. Live vaccination is useful in preventing colonisation of mucosal-dwelling pathogens by inducing a mucosal immune response. Native poultry isolate Ligilactobacillus agilis La3 (previously Lactobacillus agilis) has been identified as a candidate for use as a live vector to deliver therapeutic proteins such as bacteriocins, phage endolysins, or vaccine antigens to the gastrointestinal tract of chickens. In this study, the complete genome sequence of L. agilis La3 was determined and transcriptome analysis was undertaken to identify highly expressed genes. Predicted promoter regions and ribosomal binding sites from constitutively expressed genes were used to construct recombinant protein expression cassettes. A series of double-crossover shuttle plasmids were constructed, to facilitate rapid selectable integration of expression cassettes into the L. agilis La3 chromosome via homologous recombination. Inserts showed 100% stable integration over 100 generations without selection. A positive relationship was found between protein expression levels and the predicted strength of the promoters. Using this system, stable chromosomal expression of a Clostridium perfringens antigen, rNetB, was demonstrated without selection. Finally, two recombinant strains, L. agilis La3::Peft-rnetB and L. agilis La3::Pcwah-rnetB, were constructed, characterised, and showed potential for future application as live vaccines in chickens. Importance Therapeutic proteins such as antigens can be used to prevent infectious diseases in poultry. However, traditional vaccine delivery by intramuscular or subcutaneous injection has generally not proven effective for mucosal-dwelling microorganisms that live within the gastrointestinal tract. Utilising live bacteria to deliver vaccine antigens directly to the gut immune system can overcome some of the limitations of conventional vaccination. In this work, Ligilactobacillus agilis La3, an especially effective gut coloniser has been analysed and engineered with modular and stable expression systems to produce recombinant proteins. To demonstrate the effectiveness of the system, expression of a vaccine antigen from poultry pathogen Clostridium perfringens was monitored over 100 generations without selection and found to be completely stable. This study demonstrates the development of genetic tools, novel constitutive expression systems and further development of L. agilis La3 as a live delivery vehicle for recombinant proteins.

Comparison of α2,6-sialyltransferases for sialylation of therapeutic proteins

Glycobiology ◽  
2019 ◽  
Vol 29 (10) ◽  
pp. 735-747
Author(s):  
Bettina Janesch ◽  
Hirak Saxena ◽  
Lyann Sim ◽  
Warren W Wakarchuk
Keyword(s):  
Protein Expression ◽  
Half Life ◽  
Therapeutic Efficacy ◽  
Therapeutic Proteins ◽  
Microtiter Plate ◽  
Sialic Acids ◽  
Expression Systems

AbstractThe development of therapeutic proteins for the treatment of numerous diseases is one of the fastest growing areas of biotechnology. Therapeutic efficacy and serum half-life are particularly important, and these properties rely heavily on the glycosylation state of the protein. Expression systems to produce authentically fully glycosylated therapeutic proteins with appropriate terminal sialic acids are not yet perfected. The in vitro modification of therapeutic proteins by recombinant sialyltransferases offers a promising and elegant strategy to overcome this problem. Thus, the detailed expression and characterization of sialyltransferases for completion of the glycan chains is of great interest to the community. We identified a novel α2,6-sialyltransferase from Helicobacter cetorum and compared it to the human ST6Gal1 and a Photobacterium sp. sialyltransferase using glycoprotein substrates in a 96-well microtiter-plate-based assay. We demonstrated that the recombinant α2,6-sialyltransferase from H. cetorum is an excellent catalyst for modification of N-linked glycans of different therapeutic proteins.

scholarly journals Expression Systems and Species Used for Transgenic Animal Bioreactors

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Yanli Wang ◽  
Sihai Zhao ◽  
Liang Bai ◽  
Jianglin Fan ◽  
Enqi Liu
Keyword(s):  
Recombinant Proteins ◽  
Mammalian Cells ◽  
Mammalian Species ◽  
Therapeutic Proteins ◽  
Transgenic Animals ◽  
Mammary Glands ◽  
Transgenic Animal ◽  
High Fertility ◽  
Expression Systems ◽  
Specific Expression

Transgenic animal bioreactors can produce therapeutic proteins with high value for pharmaceutical use. In this paper, we compared different systems capable of producing therapeutic proteins (bacteria, mammalian cells, transgenic plants, and transgenic animals) and found that transgenic animals were potentially ideal bioreactors for the synthesis of pharmaceutical protein complexes. Compared with other transgenic animal expression systems (egg white, blood, urine, seminal plasma, and silkworm cocoon), the mammary glands of transgenic animals have enormous potential. Compared with other mammalian species (pig, goat, sheep, and cow) that are currently being studied as bioreactors, rabbits offer many advantages: high fertility, easy generation of transgenic founders and offspring, insensitivity to prion diseases, relatively high milk production, and no transmission of severe diseases to humans. Noticeably, for a small- or medium-sized facility, the rabbit system is ideal to produce up to 50 kg of protein per year, considering both economical and hygienic aspects; rabbits are attractive candidates for the mammary-gland-specific expression of recombinant proteins. We also reviewed recombinant proteins that have been produced by targeted expression in the mammary glands of rabbits and discussed the limitations of transgenic animal bioreactors.

scholarly journals Characterization of synthetic riboswitch in cell-free protein expression systems

RNA Biology ◽  
2021 ◽  
pp. 1-12
Author(s):  
Yaroslav Chushak ◽  
Svetlana Harbaugh ◽  
Kathryn Zimlich ◽  
Bryan Alfred ◽  
Jorge Chávez ◽  
...  
Keyword(s):  
Protein Expression ◽  
Expression Systems ◽  
Free Protein

Adaptation of protein expression byEscherichia coliin the gastrointestinal tract of gnotobiotic mice

2009 ◽  
Vol 11 (4) ◽  
pp. 751-761 ◽  
Author(s):  
Carl Alpert ◽  
Jacqueline Scheel ◽  
Wolfram Engst ◽  
Gunnar Loh ◽  
Michael Blaut
Keyword(s):  
Gastrointestinal Tract ◽  
Protein Expression ◽  
Gnotobiotic Mice

scholarly journals Quantitative Detection of Clostridium perfringens in the Broiler Fowl Gastrointestinal Tract by Real-Time PCR

2005 ◽  
Vol 71 (7) ◽  
pp. 3911-3916 ◽  
Author(s):  
Mark G. Wise ◽  
Gregory R. Siragusa
Keyword(s):  
Gastrointestinal Tract ◽  
Real Time ◽  
Clostridium Perfringens ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Quantitative Detection ◽  
Necrotic Enteritis ◽  
Analytical Sensitivity ◽  
Quantitative Real Time Pcr ◽  
Pcr Inhibitor

ABSTRACT Strains of Clostridium perfringens are a frequent cause of food-borne disease and gas gangrene and are also associated with necrotic enteritis in chickens. To detect and quantify the levels of C. perfringens in the chicken gastrointestinal tract, a quantitative real-time PCR assay utilizing a fluorogenic, hydrolysis-type probe was developed and utilized to assay material retrieved from the broiler chicken cecum and ileum. Primers and probe were selected following an alignment of 16S rDNA sequences from members of cluster I of the genus Clostridium, and proved to be specific for C. perfringens. The assay could detect approximately 50 fg of C. perfringens genomic DNA and approximately 20 cells in pure culture. Measurements of the analytical sensitivity determined with spiked intestinal contents indicated that the consistent limit of detection with ileal samples was approximately 102 CFU/g of ileal material, but only about 104 CFU/g of cecal samples. The decreased sensitivity with the cecal samples was due to the presence of an unidentified chemical PCR inhibitor(s) in the cecal DNA purifications. The assay was utilized to rapidly detect and quantify C. perfringens levels in the gut tract of broiler chickens reared without supplementary growth-promoting antibiotics that manifested symptoms of necrotic enteritis. The results illustrated that quantitative real-time PCR correlates well with quantification via standard plate counts in samples taken from the ileal region of the gastrointestinal tract.

scholarly journals An overview on current molecular tools for heterologous gene expression in Trichoderma

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Irene Tomico-Cuenca ◽  
Robert L. Mach ◽  
Astrid R. Mach-Aigner ◽  
Christian Derntl
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Protein Secretion ◽  
Heterologous Gene Expression ◽  
Heterologous Gene ◽  
Molecular Tools ◽  
Expression Systems ◽  
Industrial Enzymes ◽  
Nutrient Sources ◽  
Transformation Methods

AbstractFungi of the genus Trichoderma are routinely used as biocontrol agents and for the production of industrial enzymes. Trichoderma spp. are interesting hosts for heterologous gene expression because their saprotrophic and mycoparasitic lifestyles enable them to thrive on a large number of nutrient sources and some members of this genus are generally recognized as safe (GRAS status). In this review, we summarize and discuss several aspects involved in heterologous gene expression in Trichoderma, including transformation methods, genome editing strategies, native and synthetic expression systems and implications of protein secretion. This review focuses on the industrial workhorse Trichoderma reesei because this fungus is the best-studied member of this genus for protein expression and secretion. However, the discussed strategies and tools can be expected to be transferable to other Trichoderma species.

scholarly journals The multifunctionality of expression systems in Bacillus subtilis: Emerging devices for the production of recombinant proteins

2021 ◽  
pp. 153537022110301
Author(s):  
Caio Coutinho de Souza ◽  
Jander Matos Guimarães ◽  
Soraya dos Santos Pereira ◽  
Luis André Morais Mariúba
Keyword(s):  
Bacillus Subtilis ◽  
Recombinant Proteins ◽  
Large Scale ◽  
Recombinant Expression ◽  
Academic Research ◽  
Future Research ◽  
Expression Systems ◽  
Bioactive Substances ◽  
Exogenous Dna ◽  
Chemical Inducers

Bacillus subtilis is a successful host for producing recombinant proteins. Its GRAS (generally recognized as safe) status and its remarkable innate ability to absorb and incorporate exogenous DNA into its genome make this organism an ideal platform for the heterologous expression of bioactive substances. The factors that corroborate its value can be attributed to the scientific knowledge obtained from decades of study regarding its biology that has fostered the development of several genetic engineering strategies, such as the use of different plasmids, engineering of constitutive or double promoters, chemical inducers, systems of self-inducing expression with or without a secretion system that uses a signal peptide, and so on. Tools that enrich the technological arsenal of this expression platform improve the efficiency and reduce the costs of production of proteins of biotechnological importance. Therefore, this review aims to highlight the major advances involving recombinant expression systems developed in B. subtilis, thus sustaining the generation of knowledge and its application in future research. It was verified that this bacterium is a model in constant demand and studies of the expression of recombinant proteins on a large scale are increasing in number. As such, it represents a powerful bacterial host for academic research and industrial purposes.

Sign in / Sign up

Export Citation Format

Share Document