High level expression, purification and immunogenicity analysis of a protective recombinant protein against botulinum neurotoxin type E

Valipour, Ebrahim; Moosavi, Mir-Latif; Amani, Jafar; Nazarian, Javad

doi:10.1007/s11274-014-1609-0

Cited by 7 publications

(7 citation statements)

References 30 publications

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“…The purity of the plasmid DNA was controlled by agarose gel electrophoresis and U.V spectroscopy. DNA concentration is estimated by measuring the absorbance at 260 nm (A260 = 1.0 for 50 μg/mL) [18].…”

Section: Preparation Of Dnamentioning

confidence: 99%

Study of DNA-Binding Activity and Antibacterial Effect of Escitalopram Oxalate, an Extensively Prescribed Antidepressant

2019

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Escitalopram oxalate (EO) is considered as one of the extensively prescribed antidepressant drug in Turkey and some other countries, therefore this research was aimed to study the interaction of the drug with DNA and study of the substance effect on bacterial growth. The absorption value of the drug solution at 238 nm was increased when DNA was added gradually to it and it showed hyperchromism effect. The value obtained for DNA binding constant (Kb) was 0.035 M −1. When we added the CuCl2 2H2O to the mixture, any breakage was not shown in double strand DNA in comparison with control DNA. In addition low concentration of EO couldn’t protect DNA (0.5273 µmole bp) against Hydroxyl free radical (0.12 µmole) although it could protect the DNA when it was at the same or higher concentrations (0.5273, 5.273 and 252.73 µmole) than the DNA concentration. In addition, MIC of the drug for E.coli and Bacillus subtilis was almost 0.185 mM and 0.55 mM respectively. The E.coli strain was killed at concentrations 45, 15, 5 mM while the Bacillus subtilis was stable against all of the concentrations.

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Section: Preparation Of Dnamentioning

confidence: 99%

Study of DNA-Binding Activity and Antibacterial Effect of Escitalopram Oxalate, an Extensively Prescribed Antidepressant

2019

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“…The expression and purification of rtUlp1, and the cleavage of the SUMO fusion protein were evaluated under reducing conditions by SDS-PAGE (12 %) or tricine/SDS-PAGE (16 %) after staining with Coomassie Blue R-250 [12]. The identification of the rtUlp1 were conducted by western blot as previously described [27,28] using anti-6 9 His tags mouse antibody (1:2500) and goat anti-mouse HRP-conjugated IgG antibody (1:10000) by enhanced chemiluminescence (ECL, Pierce, Thermo Fisher Scientific).…”

Section: Sds-page and Western Blotmentioning

confidence: 99%

A Novel Strategy for the Preparation of Codon-Optimized Truncated Ulp1 and its Simplified Application to Cleavage the SUMO Fusion Protein

Wang

Liu

et al. 2016

Protein J

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Ubiquitin-like protease 1 (Ulp1) of Saccharomyces cerevisiae emerges as a fundamental tool to obtain the natural N-terminal target protein by cleavage of the small ubiquitin-related modifier (SUMO) fusion protein. However, the costly commercial Ulp1 and its complicated procedures limit its application in the preparation of the target protein with natural N-terminal sequence. Here, we describe the preparation of bioactive codon-optimized recombinant truncated Ulp1 (Leu403-Lys621) (rtUlp1) of S. cerevisiae in Escherichia coli using only one-step with Ni-NTA affinity chromatograph, and the application of rtUlp1 to cleave the SUMO fusion protein by simply mixing the purified rtUlp1, SUMO fusion protein and DL-Dithiothreitol in Tris-HCl buffer. The optimal expression level of non-fusion protein rtUlp1 accounts for approximately 50 % of the total cellular protein and 36% of the soluble form by addition of isopropyl β-D-l-thiogalactopyranoside at a final concentration of 0.4 mM at 18 °C for 20 h. The purification of target protein rtUlp1 was conducted by Ni-NTA affinity chromatography. The final yield of rtUlp1 was 45 mg/l in flask fermentation with a purity up to 95%. Furthermore, the high purity of rtUlp1 could effectively cleave the SUMO-tTβRII fusion protein (SUMO gene fused to truncated transforming growth factor-beta receptor type II gene) with the above simplified approach, and the specific activity of the rtUlp1 reached up to 2.8 × 10(4) U/mg, which is comparable to the commercial Ulp1. The preparation and application strategy of the rtUlp1 with commonly available laboratory resources in this study will be convenient to the cleavage of the SUMO fusion protein to obtain the natural N-terminal target protein, which can be implemented in difficult-to-express protein functional analysis.

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“…The samples mentioned above, were mixed with equivalent sample buffer (125 mM Tris-HCl, pH 6.8, 20 % glycerol, 4 % SDS, 0.005 % bromophenol blue, and 10 % 2-mercaptoethanol). The proteins were detected by Coomassie brilliant blue R-250 staining or transferred onto a nitrocellulose membrane for Western blot analysis (Liu et al 2011;Valipour et al 2014). …”

Section: Sds-page and Western Blotmentioning

confidence: 99%

High-level expression and characterization of bioactive human truncated variant of hepatocyte growth factor in Escherichia coli

Wang

Liu

Zhang

et al. 2014

World J Microbiol Biotechnol

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Hepatocyte growth factor (HGF) is an effective anti-fibrotic factor because of its bioactivity in inhibiting fibrosis-related proteins in the development of hepatic fibrosis. However, high-level production of bioactive mature form HGF is difficult because of its complex structure. Here, we report a non-fusion protein expression system to obtain truncated variant of N-terminal hairpin and first kringle domains of HGF (tvNK1) in Escherichia coli to determine its anti-fibrotic effects on hepatic stellate cells (HSCs). Under the selected conditions of cultivation and isopropyl-β-D-1-thiogalactopyranoside induction, the expression level of tvNK1 accounted for approximately 65 % of the total cellular protein and 50 % of fusion protein in the supernatant of whole cell lysates. The recombinant protein could be purified in one step with Ni(2+)-affinity chromatograph. Finally, about 65 mg recombinant tvNK1 was obtained from 1 l fermentation culture with no <95 % purity. In vitro, the final purified tvNK1 was shown to inhibit the proliferation of HSCs and decrease the mRNA and protein expression levels of fibrosis-related COL1A1 and α-smooth muscle actin genes.

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High level expression, purification and immunogenicity analysis of a protective recombinant protein against botulinum neurotoxin type E

Cited by 7 publications

References 30 publications

Study of DNA-Binding Activity and Antibacterial Effect of Escitalopram Oxalate, an Extensively Prescribed Antidepressant

Study of DNA-Binding Activity and Antibacterial Effect of Escitalopram Oxalate, an Extensively Prescribed Antidepressant

A Novel Strategy for the Preparation of Codon-Optimized Truncated Ulp1 and its Simplified Application to Cleavage the SUMO Fusion Protein

High-level expression and characterization of bioactive human truncated variant of hepatocyte growth factor in Escherichia coli

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