High level expression of recombinant BoNT/A-Hc by high cell density cultivation of Escherichia coli

Yari, Kheirollah; Fatemi, Seyed Safa-Ali; Tavallaei, Mahmood

doi:10.1007/s00449-011-0579-y

Cited by 15 publications

(5 citation statements)

References 22 publications

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“…Most studies using E. coli to produce recombinant H C domains yield an average of 40 mg/L, except when a production optimization procedure is performed [30]. When only H C C and H C D production is considered, the yield average is higher, reaching 70 mg/L-culture [13,18,31,32].…”

Section: Discussionmentioning

confidence: 99%

Production and Evaluation of a Recombinant Chimeric Vaccine against Clostridium botulinum Neurotoxin Types C and D

et al. 2013

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Bovine botulism is a fatal disease that is caused by botulinum neurotoxins (BoNTs) produced by Clostridium botulinum serotypes C and D and that causes great economic losses, with nearly 100% lethality during outbreaks. It has also been considered a potential source of human food-borne illness in many countries. Vaccination has been reported to be the most effective way to control bovine botulism. However, the commercially available toxoid-based vaccines are difficult and hazardous to produce. Neutralizing antibodies targeted against the C-terminal fragment of the BoNT heavy chain (HC) are known to confer efficient protection against lethal doses of BoNTs. In this study, a novel recombinant chimera, consisting of Escherichia coli heat-labile enterotoxin B subunit (LTB), a strong adjuvant of the humoral immune response, fused to the HC of BoNT serotypes C and D, was produced in E. coli. Mice vaccinated with the chimera containing LTB and an equivalent molar ratio of the chimera without LTB plus aluminum hydroxide (Al(OH)3) developed 2 IU/mL of antitoxins for both serotypes. Guinea pigs immunized with the recombinant chimera with LTB plus Al(OH)3 developed a protective immune response against both BoNT/C (5 IU/mL) and BoNT/D (10 IU/mL), as determined by a mouse neutralization bioassay with pooled sera. The results achieved with guinea pig sera fulfilled the requirements of commercial vaccines for prevention of botulism, as determined by the Brazilian Ministry of Agriculture, Livestock and Food, Supply. The presence of LTB was essential for the development of a strong humoral immune response, as it acted in synergism with Al(OH)3. Thus, the vaccine described in this study is a strong candidate for the control of botulism in cattle.

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Section: Discussionmentioning

confidence: 99%

Production and Evaluation of a Recombinant Chimeric Vaccine against Clostridium botulinum Neurotoxin Types C and D

et al. 2013

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“…After 12 hours of batch culture, adding yeast extract and glucose resulted in higher protein content (per gram of cell mass), and then fed batch techniques were used to achieve high cell densities [ 36 ]. Fed-batch systems produced a high cell density and increased recombinant protein production by 4–8 folds [ 10 37 ]. The relationship between growth rates in time and change in biomass can easily be noticed as the exponential growth period between 12 and 22 hours is the most remarkable.…”

Section: Discussionmentioning

confidence: 99%

Expression of full and fragment-B of diphtheria toxin genes inEscherichia colifor generating of recombinant diphtheria vaccines

Abulmagd

Khattab

Zedan

2022

Clin Exp Vaccine Res

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Purpose In the present study, whole diphtheria toxin (dt) and fragment B (dtb) genes from Corynebacterium diphtheriae Park William were cloned into Escherichia coli , the purified expressed proteins were evaluated for ultimately using as a candidate vaccine. Materials and Methods The dt and dtb genes were isolated from bacterial strain ATCC (American Type Culture Collection) no. 13812. Plasmid pET29a+ was extracted by DNA-spin TM plasmid purification kit where genes were inserted using BamHI and HindIII-HF. Cloned pET29a+dt and pET29a+dtb plasmids were transformed into E. coli BL21(DE3)PlysS as expression host. The identity of the sequences was validated by blasting the sequence (BLASTn) against all the reported nucleotide sequences in the NCBI (National Center for Biotechnology Information) GenBank. Production of proteins in high yield by different types and parameters of fermentation to determine optimal conditions. Lastly, the purified concentrated rdtx and rdtb were injected to BALB/c mice and antibody titers were detected. Results The genetic transformation of E. coli DH5α and E. coli BL21 with the pET-29a(+) carrying the dt and dtb genes was confirmed by colony polymerase chain reaction assay and were positive to grow on Luria-Bertani/kanamycin medium. The open reading frame of dt and dtb sequences consisted of 1,600 bp and 1,000 bp, were found to be 100% identical to dt and dtb sequence of C. diphtheriae (accession number KX702999.1 and KX702993.1) respectively. The optimal condition for high cell density is fed-batch fermentation production to express the rdtx and rdtb at 280 and 240 Lf/mL, dissolved oxygen was about 24% and 22% and the dry cell weight of bacteria was 2.41 g/L and 2.18 g/L, respectively. Conclusion This study concluded with success in preparing genetically modified two strains for the production of a diphtheria vaccine, and to reach ideal production conditions to achieve the highest productivity.

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“…Yari et al studied the effect of temperature on the expression of the recombinant BoNT/A-Hc in E. coli . Temperature (30 and 37 °C) did not have significant effect on the protein expression but low level of temperature favored cell cultivation (Yari et al 2010 , 2012 ).…”

Section: Discussionmentioning

confidence: 99%

Optimization of rPDT fusion protein expression by Escherichia coli in pilot scale fermentation: a statistical experimental design approach

et al. 2018

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High yield recombinant protein production is highly desirable for biotechnological purposes. In the design of recombinant expression conditions, a number of essential central elements such as expression strain, type of medium, bioprocess optimization, and mathematical modeling should be considered. Well-designed industrial scale production of one recombinant protein with optimized influential parameters and yield can address the cost and production reproducibility issues. In the present study, statistical experimental design methodology was used to investigate the effect of fermentation conditions (dissolved oxygen, IPTG, and temperature) on rPDT production by Escherichia coli. rPDT is a recombinant fusion protein consisting of three different protein domains including the N-terminal 179 amino acid fragment of the S1 subunit of pertussis toxin, the full-length genetically detoxified diphtheria toxin (CRM197), and the 50 kDa tetanus toxin fragment C. A 15 Box–Behnken design augmented with center points revealed that IPTG and DO at the center point and low temperature will result in high yield. The optimal condition for rPDT production were found to be 100 µM IPTG, DO 30% and temperature 20 °C.

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High level expression of recombinant BoNT/A-Hc by high cell density cultivation of Escherichia coli

Cited by 15 publications

References 22 publications

Production and Evaluation of a Recombinant Chimeric Vaccine against Clostridium botulinum Neurotoxin Types C and D

Production and Evaluation of a Recombinant Chimeric Vaccine against Clostridium botulinum Neurotoxin Types C and D

Expression of full and fragment-B of diphtheria toxin genes inEscherichia colifor generating of recombinant diphtheria vaccines

Optimization of rPDT fusion protein expression by Escherichia coli in pilot scale fermentation: a statistical experimental design approach

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