Novel plasmid-based expression vectors for intra- and extracellular production of recombinant proteins in Bacillus subtilis

Phan, Trang Thi Phuong; Nguyen, Hoang Duc; Schumann, Wolfgang

doi:10.1016/j.pep.2005.07.005

Cited by 105 publications

(82 citation statements)

References 26 publications

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“…1a). The vaccine vector pLDV702 was constructed after cloning the spaP1N gene (1.4 kb), which encodes the P1 antigen from S. mutans strain UA159 (29), into the pHCMC03 vector under the control of PgsiB, which is active only during the vegetative growth stage (9,30) (Fig. 1b).…”

Section: Methodsmentioning

confidence: 99%

“…P1 expression by the B. subtilis vaccine strains was monitored in vitro after induction by high-temperature incubation (42°C for 4 h), as previously described (30). Briefly, cells cultivated in LB broth at 37°C were shifted to 42°C at an optical density at 600 nm (OD 600 ) of 0.6 to 0.8 and incubated for an additional 4 h. The cells were collected and suspended in lysis buffer (30% sucrose, 100 mM Tris HCl, pH 7.2, 800 g/ml of lysozyme) for 30 min at 37°C, with SDS (final concentration, 1%) added to lyse the cells.…”

Section: Methodsmentioning

confidence: 99%

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Gut Adhesive Bacillus subtilis Spores as a Platform for Mucosal Delivery of Antigens

et al. 2014

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Bacterial carriers for the mucosal delivery of antigens, particularly those capable of colonizing or invading through the mucosal epithelia, have been intensively investigated (1). Live bacterial vectors can be generated with attenuated pathogens, such as Salmonella and Listeria, or nonpathogenic commensal species, such as Lactococcus and Lactobacillus. Bacteria in the former group of are capable of inducing strong and long-lasting immune responses to passenger antigens but present serious safety concerns, whereas bacteria in the latter group are safer but typically induce much lower immune responses to the passenger antigens (1-3). As a safe nonpathogenic live bacterial vector, Bacillus subtilis, a spore-forming soil Gram-positive bacterial species, has been engineered to express antigens as either vegetative cells or spores (4, 5). As antigen carriers, B. subtilis spores have several attractive features, including a safe record of human and animal use as both probiotic and food additives, remarkable heat resistance, and rather easy genetic and bacteriological manipulation.Currently, two major genetic approaches have been proposed to generate recombinant B. subtilis spores as vaccine delivery vectors. The first approach relies on the expression of a heterologous protein genetically fused to surface-exposed spore coat proteins, such as CotB, CotC, or CotG (6, 7). Such a strategy would allow a better presentation of the passenger antigen to the mucosa-associated lymphoid tissue (MALT) afferent sites, leading to the induction of adaptive immune responses, such as mucosal secretory (IgA) or systemic (IgG) antigen-specific antibody responses (6-8). The second approach is based on a distinct rationale and has employed episomal vectors in which the target antigen is expressed under the control of a promoter (PgsiB) active only at the vegetative cell stage, which means immediately after spore germination (9-11). This antigen delivery approach relies on the fact that B. subtilis spores germinate during transit through the gastrointestinal tract and produce the target antigen at the intestinal lumen or inside the phagocytes of antigen-presenting cells (APCs), leading to the induction of antibody responses in the serum (IgG) and mucosa (fecal IgA) (9-11).However, in both cases, the administration of recombinant spores via mucosal routes typically confers immune responses to the passenger antigen lower than those achieved with delivery systems based on attenuated bacterial strains capable of colonizing the mammalian gastrointestinal tract. The reduced mucosal adjuvant effects of B. subtilis spores have been attributed to several factors, such as a previously established immunity generated by the frequent ingestion of spores, the reduced amount of expressed antigens, and the rapid transit through the gastrointestinal tract, which reduces the chances of a productive interaction with the gut-associated lymphoid tissue (GALT), such as M cells and APCs at Peyer's patches (PPs) (12, 13).In an attempt to improve the performance o...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Gut Adhesive Bacillus subtilis Spores as a Platform for Mucosal Delivery of Antigens

et al. 2014

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“…Priority was given to the development of a novel, technically compliant bacterial gene expression system that would be both cheap and highly reliable. Regulated promoters used in expression vectors can be induced by sugars, isopropyl-␤-D-thiogalactopyranoside (IPTG), temperature shifts, acid shock, or ethanol (27,28,29,35). If such systems are to be technically exploited, ways need to be found to circumvent the high price of inducers required.…”

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confidence: 99%

Self-Inducible Bacillus subtilis Expression System for Reliable and Inexpensive Protein Production by High-Cell-Density Fermentation

Wenzel

Muller

Siemann‐Herzberg

et al. 2011

Appl Environ Microbiol

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A novel technically compliant expression system was developed for heterologous protein production in Bacillus subtilis with the aim of increasing product yields at the same time as decreasing production costs. Standard systems involve the positively regulated manP promoter of the mannose operon, which led to relatively high product yields of 5.3% (5.3 g enhanced green fluorescent protein [eGFP] per 100 g cell dry weight [CDW]) but required large quantities of mannose to induce the reactions, thus rendering the system's technical application rather expensive. To improve this situation, mutant B. subtilis strains were used: the ⌬manA (mannose metabolism) strain TQ281 and the ⌬manP (mannose uptake) strain TQ356. The total amount of inducer could be reduced with TQ281, which, however, displayed sensitivity to mannose. An inducer-independent self-induction system was developed with TQ356 to further improve the cost efficiency and product yield of the system, in which glucose prevents induction by carbon catabolite repression. To create optimal self-induction conditions, a glucose-limited process strategy, namely, a fed-batch process, was utilized as follows. The initiation of self-induction at the beginning of the glucose-restricted transition phase between the batch and fed-batch phase of fermentation and its maintenance throughout the glucose-limiting fed-batch phase led to a nearly 3-fold increase of product yield, to 14.6%. The novel B. subtilis self-induction system thus makes a considerable contribution to improving product yield and reducing the costs associated with its technical application.The Gram-positive soil bacterium Bacillus subtilis is an attractive host for the production of various industrial proteins, such as amylases, proteases, and lipases. It is classified as a generally recognized as safe (GRAS) organism due to its lack of pathogenicity and absence of endotoxins. Further advantages of B. subtilis are its direct secretion of proteins into the medium via the Sec and Tat protein secretion machineries (40), capacity for genetic manipulation, easy handling, short processing times, and application in large-scale industrial production of proteins, for example, laundry enzymes and riboflavin (34). Homologous proteins can be produced in the order of several grams per liter, but only a few milligrams per liter of proteins can be produced from heterologous sources (34,35). When it comes to the biotechnological production of heterologous proteins, care must be taken to enable the reliable and inexpensive production of high yields of desired proteins. Priority was given to the development of a novel, technically compliant bacterial gene expression system that would be both cheap and highly reliable. Regulated promoters used in expression vectors can be induced by sugars, isopropyl-␤-D-thiogalactopyranoside (IPTG), temperature shifts, acid shock, or ethanol (27,28,29,35). If such systems are to be technically exploited, ways need to be found to circumvent the high price of inducers required. Autoinducible s...

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“…These results indicate that 3-OST-1 collected from E. coli contains approximately 4×10 6 endotoxin units (EU) per mL. The Bacillus-expressed enzymes have drastically lower endotoxin unit per milliliter levels.…”

Section: Endotoxin Levels Of Expressed Enzymementioning

confidence: 85%

“…However, production of high-value eukaryotic proteins for pharmaceutical applications remains a major challenge in B. subtilis. One problem with the production of eukaryotic proteins is the absence of expression or relatively low expression efficiency [6]. Overproduction of recombinant proteins in B. subtilis is a complex process requiring the design of improved production strains and a comprehensive understanding of the cellular physiology and protein expression mechanisms.…”

Section: Introductionmentioning

confidence: 99%

Expression of Low Endotoxin 3-O-Sulfotransferase in Bacillus subtilis and Bacillus megaterium

Wang

Englaender

et al. 2013

Appl Biochem Biotechnol

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A key enzyme for the biosynthesis and bioengineering of heparin, 3-O-sulfotransferase-1 (3-OST-1), was expressed and purified in Gram-positive Bacillus subtilis and Bacillus megaterium. Western blotting, protein sequence analysis, and enzyme activity measurement confirmed the expression. The enzymatic activity of 3-OST-1 expressed in Bacillus species were found to be similar to those found when expressed in Escherichia coli. The endotoxin level in 3-OST-1 from B. subtilis and B. megaterium -fold lower than that of the E. coli-expressed 3-OST-1, which makes the Bacillus expression system of particular interest for the generation of pharmaceutical grade raw heparin from nonanimal sources.

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Novel plasmid-based expression vectors for intra- and extracellular production of recombinant proteins in Bacillus subtilis

Cited by 105 publications

References 26 publications

Gut Adhesive Bacillus subtilis Spores as a Platform for Mucosal Delivery of Antigens

Gut Adhesive Bacillus subtilis Spores as a Platform for Mucosal Delivery of Antigens

Self-Inducible Bacillus subtilis Expression System for Reliable and Inexpensive Protein Production by High-Cell-Density Fermentation

Expression of Low Endotoxin 3-O-Sulfotransferase in Bacillus subtilis and Bacillus megaterium

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