A novel β-galactosidase gene (PbBgal35A) from Pedobacter sp. CAUYN2 was cloned and expressed in Escherichia coli. The gene had an open reading frame of 1917 bp, encoding 638 amino acids with a predicted molecular mass of 62.3 kDa. The deduced amino acid sequence of the gene shared the highest identity of 41% with a glycoside hydrolase family 35 β-galactosidase from Xanthomonas campestris pv. campestris (AAP86763.1). The recombinant β-galactosidase (PbBgal35A) was purified to homogeneity with a specific activity of 65.9 U/mg. PbBgal35A was optimally active at pH 5.0 and 50 °C, respectively, and it was stable within pH 4.5‒7.0 and up to 45 °C. PbBgal35A efficiently synthesized galacto-oligosaccharides from lactose with a conversion ratio of 32% (w/w) and fructosyl-galacto-oligosaccharides from lactulose with a conversion ratio of 21.9% (w/w). Moreover, the enzyme catalyzed the synthesis of galacto-oligosaccharides from low-content lactose in fresh milk, and the GOS conversion ratios of 17.1% (w/w) and 7.8% (w/w) were obtained when the reactions were performed at 45 and 4 °C, respectively. These properties make PbBgal35A an ideal candidate for commercial use in the manufacturing of GOS-enriched dairy products.
- MeSH
- bakteriální proteiny genetika metabolismus chemie MeSH
- beta-galaktosidasa * genetika metabolismus chemie izolace a purifikace MeSH
- Escherichia coli genetika metabolismus MeSH
- exprese genu MeSH
- glykosylace MeSH
- klonování DNA * MeSH
- koncentrace vodíkových iontů MeSH
- laktosa * metabolismus MeSH
- mléko mikrobiologie MeSH
- molekulová hmotnost MeSH
- oligosacharidy metabolismus MeSH
- Pedobacter * enzymologie genetika MeSH
- rekombinantní proteiny genetika metabolismus chemie izolace a purifikace MeSH
- sekvence aminokyselin MeSH
- stabilita enzymů * MeSH
- substrátová specifita MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
Lipases are industrially important enzymes having vast applications in various fields. Cloning and expression of lipase enzyme-encoding genes in suitable host lead to their widespread use in different fields. The present study represents the first attempt towards the expression of the synthetic lipase gene in Pseudomonas aeruginosa. An alkalophilic lipase gene (GenBank accession number: NP_388152) from Bacillus subtilis was synthetically designed and introduced in the pJN105 vector and subsequently cloned in Pseudomonas aeruginosa SDK-6. Agarose gel electrophoresis confirmed the transformation of SDK-6, exhibiting a band difference of ~ 700 bp between native and recombinant pJN105. Further amplification of cloned lipase gene was confirmed using PCR amplification with Lip 1 and Lip 2 primers respectively, followed by restriction analysis. Approximately 15-fold increase in lipase production was observed in recombinant Pseudomonas as compared to the native strain. One factor at a time (OFAT) analysis revealed L-arabinose, inoculum size (0.5%; v/v), and agitation (120 rpm) as significant factors affecting the over-expression of lipase enzyme. Optimization of enzyme induction conditions by central composite design (CCD) led to 1.60-fold increase in the production of lipase at 0.65% (w/v) inducer concentration, OD600-1.075 before induction and 35 °C post induction temperature with overall lipase production of 50.50 IU/mL. Statistical validation of observed value via ANOVA showed an F-value of 138.70 at p < 0.01 with R2 of 0.9921.
- MeSH
- arabinosa metabolismus MeSH
- Bacillus subtilis * genetika enzymologie MeSH
- bakteriální proteiny genetika metabolismus MeSH
- exprese genu MeSH
- genetické vektory genetika MeSH
- klonování DNA MeSH
- lipasa * genetika metabolismus MeSH
- Pseudomonas aeruginosa * genetika enzymologie MeSH
- rekombinantní proteiny * genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
L-asparaginase is an essential enzyme used in cancer treatment, but its production faces challenges like low yield, high cost, and immunogenicity. Recombinant production is a promising method to overcome these limitations. In this study, response surface methodology (RSM) was used to optimize the production of L-asparaginase 1 from Saccharomyces cerevisiae in Escherichia coli K-12 BW25113. The Box-Behnken design (BBD) was utilized for the RSM modeling, and a total of 29 experiments were conducted. These experiments aimed to examine the impact of different factors, including the concentration of isopropyl-b-LD-thiogalactopyranoside (IPTG), the cell density prior to induction, the duration of induction, and the temperature, on the expression level of L-asparaginase 1. The results revealed that while the post-induction temperature, cell density at induction time, and post-induction time all had a significant influence on the response, the post-induction time exhibited the greatest effect. The optimized conditions (induction at cell density 0.8 with 0.7 mM IPTG for 4 h at 30 °C) resulted in a significant amount of L-asparaginase with a titer of 93.52 μg/mL, which was consistent with the model-based prediction. The study concluded that RSM optimization effectively increased the production of L-asparaginase 1 in E. coli, which could have the potential for large-scale fermentation. Further research can explore using other host cells, optimizing the fermentation process, and examining the effect of other variables to increase production.
- MeSH
- asparaginasa * genetika biosyntéza metabolismus MeSH
- Escherichia coli K12 genetika enzymologie MeSH
- Escherichia coli genetika metabolismus MeSH
- fermentace MeSH
- isopropylthiogalaktosid farmakologie MeSH
- rekombinantní proteiny * genetika metabolismus MeSH
- Saccharomyces cerevisiae * genetika metabolismus MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
Pichia pastoris, a methylotrophic yeast, is known to be an efficient host for heterologous proteins production. In this study, a recombinant P. pastoris Y11430 was found better for β-glucosidase activity in comparison with a wild type P. pastoris Y11430 strain, and thereby, subjected to methanol intermittent feed profiling for β-glucosidase production. The results showed that at 72 h of cultivation time, the cultures with 16.67% and 33.33% methanol feeding with constant rate could produce the total dry cell weight of 52.23 and 118.55 g/L, respectively, while the total mutant β-glucosidase activities were 1001.59 and 3259.82 units, respectively. The methanol feeding profile was kept at 33% with three methanol feeding strategies such as constant feed rate, linear feed rate, and exponential feed rate which were used in fed-batch fermentation. At 60 h of cultivation, the highest total mutant β-glucosidase activity was 2971.85 units for exponential feed rate culture. On the other hand, total mutant β-glucosidase activity of the constant feed rate culture and linear feed rate culture were 1682.25 and 1975.43 units, respectively. The kinetic parameters of exponential feed rate culture were specific growth rate on glycerol 0.228/h, specific growth of methanol 0.061/h, maximum total dry cell weight 196.73 g, yield coefficient biomass per methanol ([Formula: see text]) 0.57 gcell/gMeOH, methanol consumption rate ([Formula: see text]) 5.76 gMeOH/h, and enzyme productivity ([Formula: see text]) 75.96 units/h. In conclusion, higher cell mass and β- glucosidase activity were produced under exponential feed rate than constant and linear feed rates.
West Nile virus (WNV) is transmitted to humans and animals by a mosquito and enters the central nervous system, leading to lethal encephalitis. Reporter viruses expressing fluorescent proteins enable detection of infected cells in vitro and in vivo, facilitating evaluation of the dynamics of viral infection, and the development of diagnostic or therapeutic methods. In this study, we developed a method for production of a recombinant replication-competent WNV expressing mCherry fluorescent protein. The expression of mCherry was observed in viral antigen-positive cells in vitro and in vivo, but the growth of the reporter WNV was reduced as compared to the parental WNV. The expression of mCherry was stable during 5 passages in reporter WNV-infected culture cells. Neurological symptoms were observed in mice inoculated intracranially with the reporter WNV. The reporter WNV expressing mCherry will facilitate research into WNV replication in mouse brains.
- MeSH
- lidé MeSH
- myši MeSH
- rekombinantní proteiny genetika MeSH
- virus západního Nilu * genetika MeSH
- západonilská horečka * veterinární MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cryptosporidium parvum infects enterocytes in diverse vertebrates, including humans, and causes diarrheal illness. However, no effective drugs are available for this protozoan infection. The P23 protein of C. parvum is a protective antigen, considered a potential candidate for developing an effective vaccine against cryptosporidiosis. In this study, the complementary DNA (cDNA) of the p23 gene was subcloned to Escherichia coli DH5α, with one nucleotide difference. The constructed plasmid pNZ8149-P23 was transferred by electroporation to Lactococcus lactis NZ3900, and the recombinant L. lactis NZ3900/pNZ8149-P23 strain was screened in Elliker-medium by adding bromocresolpurple indicator. A 23-kDa protein was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after nisin induction in LM17 broth medium, suggesting that P23 protein was in the form of glycosylation. Simultaneously, an optimal induction time of 9 h was determined, and the density of OD600 = 2.7 was tested. Through western blot and indirect immunofluorescence (IIF) analysis, the immunocompetence of expressed P23 antigen was identified, and its location of release to the cell interior of recombinant L. lactis was manifested. The first report of a food-grade genetically engineered L. lactis strain expressing a P23 antigen of C. parvum is herein presented. This result provides a novel and safe utilization method of P23 against C. parvum infection.
- MeSH
- Cryptosporidium parvum * genetika metabolismus MeSH
- Cryptosporidium * metabolismus MeSH
- kryptosporidióza * prevence a kontrola MeSH
- Lactococcus lactis * genetika metabolismus MeSH
- lidé MeSH
- pyridinolkarbamát MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Fructooligosaccharides (FOS) are compounds possessing various health properties and are added to functional foods as prebiotics. The commercial production of FOS is done through the enzymatic transfructolysation of sucrose by β-fructofuranosidases which is found in various organisms of which Aureobasidium pullulans and Aspergillus niger are the most well known. This study overexpressed two differently codon-optimized variations of the Aspergillus fijiensis β-fructofuranosidase-encoding gene (fopA) under the transcriptional control of either the alcohol oxidase (AOX1) or glyceraldehyde-3-phosphate dehydrogenase (GAP) promoters. When cultivated in shake flasks, the two codon-optimized variants displayed similar volumetric enzyme activities when expressed under control of the same promoter with the GAP strains producing 11.7 U/ml and 12.7 U/ml, respectively, and the AOX1 strains 95.8 U/ml and 98.6 U/ml, respectively. However, the highest production levels were achieved for both codon-optimized genes when expressed under control of the AOX1 promoter. The AOX1 promoter was superior to the GAP promoter in bioreactor cultivations for both codon-optimized genes with 13,702 U/ml and 2718 U/ml for the AOX1 promoter for ATUM and GeneArt®, respectively, and 6057 U/ml and 1790 U/ml for the GAP promoter for ATUM and GeneArt®, respectively. The ATUM-optimized gene produced higher enzyme activities when compared to the one from GeneArt®, under the control of both promoters.
The initial activation step in the gating of ubiquitously expressed Orai1 calcium (Ca2+) ion channels represents the activation of the Ca2+-sensor protein STIM1 upon Ca2+ store depletion of the endoplasmic reticulum. Previous studies using constitutively active Orai1 mutants gave rise to, but did not directly test, the hypothesis that STIM1-mediated Orai1 pore opening is accompanied by a global conformational change of all Orai transmembrane domain (TM) helices within the channel complex. We prove that a local conformational change spreads omnidirectionally within the Orai1 complex. Our results demonstrate that these locally induced global, opening-permissive TM motions are indispensable for pore opening and require clearance of a series of Orai1 gating checkpoints. We discovered these gating checkpoints in the middle and cytosolic extended TM domain regions. Our findings are based on a library of double point mutants that contain each one loss-of-function with one gain-of-function point mutation in a series of possible combinations. We demonstrated that an array of loss-of-function mutations are dominant over most gain-of-function mutations within the same as well as of an adjacent Orai subunit. We further identified inter- and intramolecular salt-bridge interactions of Orai subunits as a core element of an opening-permissive Orai channel architecture. Collectively, clearance and synergistic action of all these gating checkpoints are required to allow STIM1 coupling and Orai1 pore opening. Our results unravel novel insights in the preconditions of the unique fingerprint of CRAC channel activation, provide a valuable source for future structural resolutions, and help to understand the molecular basis of disease-causing mutations.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- fosfatidylcholiny chemie metabolismus MeSH
- gating iontového kanálu genetika MeSH
- genetické vektory chemie metabolismus MeSH
- HEK293 buňky MeSH
- interakční proteinové domény a motivy MeSH
- konformace proteinů, alfa-helix MeSH
- konformace proteinů, beta-řetězec MeSH
- lidé MeSH
- liposomy chemie metabolismus MeSH
- luminescentní proteiny genetika metabolismus MeSH
- metoda terčíkového zámku MeSH
- mutace MeSH
- nádorové proteiny chemie genetika metabolismus MeSH
- protein ORAI1 chemie genetika metabolismus MeSH
- protein STIM1 chemie genetika metabolismus MeSH
- regulace genové exprese MeSH
- rekombinantní proteiny chemie genetika metabolismus MeSH
- reportérové geny MeSH
- simulace molekulární dynamiky MeSH
- substituce aminokyselin MeSH
- vápník metabolismus MeSH
- vápníková signalizace * MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- zelené fluorescenční proteiny genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cell immunocapture microfluidic devices represent a rapidly developing field with many potential applications in medical diagnostics. The core of such approach lies in the cell binding to antibody coated surfaces through their surface receptors. Here we show, that the small recombinant protein binders (PBs) can be used for this purpose as well, with the advantage of their constructional flexibility, possibility of fusion with range of tags and cheap mass production. For this purpose, two different PBs derived from Albumin Binding Domain (ABDwt) of streptococcal protein G, so called REX and ARS ligands with proved high affinity and selectivity to the human interleukin-23 (IL-23R) and IL-17 receptor A were used. Four PBs variants recognizing two different epitopes on two different receptors and two PBs variants binding to the same epitope on one receptor but having different peptide spacer with Avitag sequence necessary for their immobilization on sensor surface were tested for cell-capture efficiency. The glass microfluidic Y-system with planar immunocapture channel working in so-called stop-flow dynamic regime was designed. Up to 60-74% immunocapture efficiency of model THP-1 cells on REX/ARS surfaces and practically no cell binding on control ABDwt surfaces was achieved. Moreover, the specific immunocapture of THP-1 cells from mixture with IL-17RA negative DU-145 cells was demonstrated. We discuss the role of the epitope, affinity and immobilization spacer of PBs as well as the influence of stop-flow dynamic regime on the effectivity of THP-1 cell immunocapture. Results can be further exploited in design of microfluidic devices for rare cells immunocapture.
- MeSH
- biosenzitivní techniky * MeSH
- lidé MeSH
- mikrofluidika MeSH
- nádorové cirkulující buňky * MeSH
- receptory interleukinu-17 MeSH
- rekombinantní proteiny genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Commensal bacterium Clostridium paraputrificum J4 produces several extracellular chitinolytic enzymes including a 62 kDa chitinase Chit62J4 active toward 4-nitrophenyl N,N'-diacetyl-β-d-chitobioside (pNGG). We characterized the crude enzyme from bacterial culture fluid, recombinant enzyme rChit62J4, and its catalytic domain rChit62J4cat. This major chitinase, securing nutrition of the bacterium in the human intestinal tract when supplied with chitin, has a pH optimum of 5.5 and processes pNGG with Km = 0.24 mM and kcat = 30.0 s-1. Sequence comparison of the amino acid sequence of Chit62J4, determined during bacterial genome sequencing, characterizes the enzyme as a family 18 glycosyl hydrolase with a four-domain structure. The catalytic domain has the typical TIM barrel structure and the accessory domains-2x Fn3/Big3 and a carbohydrate binding module-that likely supports enzyme activity on chitin fibers. The catalytic domain is highly homologous to a single-domain chitinase of Bacillus cereus NCTU2. However, the catalytic profiles significantly differ between the two enzymes despite almost identical catalytic sites. The shift of pI and pH optimum of the commensal enzyme toward acidic values compared to the soil bacterium is the likely environmental adaptation that provides C. paraputrificum J4 a competitive advantage over other commensal bacteria.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- chitin metabolismus MeSH
- chitinasy chemie genetika metabolismus MeSH
- Clostridium růst a vývoj izolace a purifikace metabolismus MeSH
- katalytická doména MeSH
- koncentrace vodíkových iontů MeSH
- lidé MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- střevní mikroflóra MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
