• Journal of Life Science
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• v.25 no.2
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• pp.130-135
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• 2015

When the alginate lyase gene (aly) from Pseudoalteromonas elyakovii IAM 14594 was expressed in E. coli, most of the gene product expressed was produced as aggregated insoluble particles known as inclusion bodies. In order to produce with an elevated level of a soluble and active form of alginate lyase in E. coli, the hyperthermophilic chaperonins (ApCpnA and ApCpnB) from archaeon Aeropyrum pernix K1 were employed as the coexpression partners. At $25^{\circ}C$ culture temperature, the level of alginate lyase activity was increased from 10.1 unit/g-soluble protein in aly single expression to 83.1 unit/g-soluble protein by coexpressing with ApCpnA and to 100.3 unit/g-soluble protein by coexpressing with ApCpnB. This results indicate that the coexpression of aly with ApCpnA and ApCpnB revealed a marked enhancement, about 8~10 fold, in the production of alginate lyase as a soluble and active form. Based on the results of various examinations on the expression variables, the optimal conditions for the maximal production of alginate lyase were determined as 1.0 mM IPTG for the inducer concentration, $25^{\circ}C$ for the culture temperature after IPTG induction, and ApCpnB for the coexpression partner. The coexpression set in the present report may be useful in the industrial production of functionally or medically important recombinant proteins in E. coli.

• Biomedical Science Letters
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• v.15 no.1
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• pp.1-8
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• 2009

Human genomic DNA is continuously attacked by oxygen radicals originated from cellular metabolic processes and numerous environmental carcinogens. 2-deoxyribonolactone (dL) is a major type of oxidized abasic (AP) lesion implicated in DNA strand scission, mutagenesis, and formation of covalent DNA-protein crosslink (DPC) with DNA polymerase (Pol) ${\beta}$. We show here that human DNA polymerase (Pol)${\iota}$ and mitochondrial $Pol{\gamma}$ give rise to stable DNA-protein crosslink (DPC) formation that is specifically mediated by dL lesion. $Pol{\gamma}$ mediates DPC formation at the incised dL residue by its 5'-deoxyribose-5-phosphate (dRP) lyase activity, while $Pol{\gamma}$ cross links with dL thorough its intrinsic dRP lyase and AP lyase activities. Reactivity in forming dL-mediated DPC was significantly higher with $Pol{\gamma}$ than with $Pol{\iota}$. DPC formation by $Pol{\gamma}$, however, can be reduced by an accessory factor of $Pol{\gamma}$ holoenzyme that may attenuate deleterious effects of crosslink adducts on mitochondrial DNA. Comparative kinetic analysis of DPC formation showed that the rate of DPC formation with either $Pol{\iota}$ or $Pol{\gamma}$ was lower than that with $Pol{\beta}$. These results revealed that the activity of catalytic lyase in DNA polymerases determine the efficiency of DPC formation with dL damages. Irreversible crosslink formation of such DNA polymerases by dL lesions may result in a prolonged strand scission and a suicide of DNA repair proteins, both of which could pose a threat to the genetic and structural integrity of DNA.

• Proceedings of the Korean Biophysical Society Conference
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• 1996.07a
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• pp.18-18
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• 1996

Bacteriophage T4 endonuclease V initiates the repair of ultraviolet (UV)-induced pyrimidine dimer photoproducts in duplex DNA. The mechanism of DNA strand cleavage involves four sequential steps: linear diffusion along dsDNA, pyrimidine dimer-specific binding, pyrimidine dimer-DNA glycosylase activity, and AP lyase activity. (omitted)

• Journal of Microbiology and Biotechnology
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• v.16 no.10
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• pp.1583-1590
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• 2006

T4 endonuclease V (T4 endo V) [EC 3. 1. 25. 1], found in bacteriophage T4, is responsible for excision repair of damaged DNA. The enzyme possesses two activities: a cyclobutane pyrimidine dimer DNA glycosylase (CPD glycosylase) and an apyrimidic/apurinic endonuclease (AP lyase). T4 denV (414 bp cDNA) encoding T4 en do V (138 amino acid) was synthesized and expressed using either an expression vector, pTriEx-4, in E. coli or a baculovirus AcNPV vector, pBacPAK8, in insect cells. The recombinant His-Tag/T4 endo V (rHis-Tag/T4 endo V) protein expressed from bacteria was purified using one-step affinity chromatography with a HiTrap Chelating HP column and used to make rabbit anti-His-Tag/T4 endo V polyclonal antibody for detection of recombinant T4 endo V (rT4 endo V) expressed in insect cells. In the meantime, the recombinant baculovirus was obtained by cotransfection of BacPAK6 viral DNA and pBP/T4 endo V in Spodoptera frugiperda (Sf21) insect cells, and used to infect Sf21 cells to overexpress T4 endo V protein. The level of rT4 endo V protein expressed in Sf21 cells was optimized by varying the virus titers and time course of infection. The optimal expression condition was set as follows; infection of the cells at a MOI of 10 and harvest at 96 h post-infection. Under these conditions, we estimated the amount of rT4 endo V produced in the baculovirus expression vector system to be 125 mg/l. The rT4 endo V was purified to homogeneity by a rapid procedure, consisting of ion-exchange, affinity, and reversed phase chromatographies, based on FPLC. The rT4 endo V positively reacted to an antiserum made against rHis-Tag/T4 endo V and showed a residual nicking activity against CPD-containing DNA caused by UV. This is the first report to have T4 endo V expressed in an insect system to exclude the toxic effect of a bacterial expression system, retaining enzymatic activity.

• Journal of Photoscience
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• v.9 no.2
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• pp.182-185
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• 2002

There are 3 UV DNA repair endonuclease activities in mammalian cells that cleave UV -irradiated DNA. Interestingly, mammalian UV endonuclease III with MW of 26.7kD has a lyase activity on AP sites. It also cleaves the phosphodiester bond within a cyclobutane pyrimidine dimer. Genomic analysis of human repair endonuclease III gene revealed that this gene has 100% sequence identity with ribosomal protein S3 (rpS3). Therefore, rpS3 seems to function both in translation and in DNA repair. This gene of about 6.1 kb contains 6 introns and 7 exons, and the first and fifth introns of human rpS3 gene contain functional U15 small nucleolar (sno) RNAs which appear to be involved in ribosome assembly. It is to be noted that the column profile of the endonuclease activity of rpS3 appears to be altered in Xeroderma Pigmentosum (XP) group D cells compared to normal cells indicating that this protein is involved in XP disease as well. XP is a human disease characterized by high sensitivity of skin by UV- or sun-light irradiation and by high frequency of developing skin cancers. We also report here that rpS3 protein is involved in other cellular functions.

• BMB Reports
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• v.38 no.6
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• pp.676-682
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• 2005

To date, no 8-oxoguanine-specific endonuclease-coding gene has been identified in Thermotoga maritima of the order Thermotogales, although its entire genome has been deciphered. However, the hypothetical protein Tm1821 from T. maritima, has a helix-hairpin-helix motif that is considered to be important for DNA binding and catalytic activity. Here, Tm1821 was overexpressed in Escherichia coli and purified using Ni-NTA affinity chromatography, protease digestion, and gel filtration. Tm1821 protein was found to efficiently cleave an oligonucleotide duplex containing 8-oxoguanine, but Tm1821 had little effect on other substrates containing modified bases. Moreover, Tm1821 strongly preferred DNA duplexes containing an 8-oxoguanine:C pair among oligonucleotide duplexes containing 8-oxoguanine paired with four different bases (A, C, G, or T). Furthermore, Tm1821 showed AP lyase activity and Schiff base formation with 8-oxoguanine in the presence of $NaBH_4$, which suggests that it is a bifunctional DNA glycosylase. Tm1821 protein shares unique conserved amino acids and substrate specificity with an 8-oxoguanine DNA glycosylase from the hyperthermophilic archaeon. Thus, the DNA recognition and catalytic mechanisms of Tm1821 protein are likely to be similar to archaeal repair protein, although T. maritima is an eubacterium.

• Journal of Microbiology and Biotechnology
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• v.15 no.3
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• pp.484-490
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• 2005

Human ribosomal protein S3 (rpS3), which has a DNA repair endonuclease activity, is a multifunctional protein. This protein is involved in DNA repair, translation, and apoptosis. In particular, rpS3 has a lyase activity, which cleaves the phosphodiester bond of damaged sites such as cyclobutane pyrimidine dimers and AP sites. Here, using deletion analysis, we identified that the repair endonuclease domain resides in the C-terminal region (165-243 aa) of rpS3. We also found that ectopic expression of GST-rpS3 in bacterial strain BL21 promoted the resistance of these cells to ultraviolet (UV) radiation and hydrogen peroxide ($H_{2}O_{2}$) treatment. The repair domain of rpS3 was sufficient to exhibit the resistance to UV irradiation and recover cell growth and viability, showing that the repair activity of rpS3 is responsible for the resistance to UV irradiation. Our study suggests that rpS3 is able to process DNA damage in bacteria via its repair domain, showing the resistance to genotoxic stress. This implies that rpS3-like activity could be operative in bacteria.

• Journal of Ginseng Research
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• v.47 no.2
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• pp.183-192
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• 2023

Viral infections are known as one of the major factors causing death. Ginseng is a medicinal plant that demonstrated a wide range of antiviral potential, and saponins are the major bioactive ingredients in the genus Panax with vast therapeutic potential. Studies focusing on the antiviral activity of the genus Panax plant-derived agents (extracts and saponins) and their mechanisms were identified and summarized, including contributions mainly from January 2016 until January 2022. P. ginseng, P. notoginseng, and P. quinquefolius were included in the review as valuable medicinal herbs against infections with 14 types of viruses. Reports from 9 extracts and 12 bioactive saponins were included, with 6 types of protopanaxadiol (PPD) ginsenosides and 6 types of protopanaxatriol (PPT) ginsenosides. The mechanisms mainly involved the inhibition of viral attachment and replication, the modulation of immune response by regulating signaling pathways, including the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, cystathionine γ-lyase (CSE)/hydrogen sulfide (H₂S) pathway, phosphoinositide-dependent kinase-1 (PDK1)/ protein kinase B (Akt) signaling pathway, c-Jun N-terminal kinase (JNK)/activator protein-1 (AP-1) pathway, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. This review includes detailed information about the mentioned antiviral effects of the genus Panax extracts and saponins in vitro and in vivo, and in human clinical trials, which provides a scientific basis for ginseng as an adjunctive therapeutic drug or nutraceutical.