• Korean Journal of Microbiology
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• v.52 no.2
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• pp.230-235
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• 2016

This study was carried out to analyze the differences in enzyme activity and stability between the native Fe superoxide dismutase (FeSOD) and the 6xHis-tagged superoxide dismutase (6xHis-FeSOD) of Streptomyces subrutilus P5. The optimum pHs for both native FeSOD and 6xHis-FeSOD were 7, while the pH range of the activity was narrower for the 6xHis-FeSOD. The native FeSOD was stable at pH 4-9, but the 6xHis-FeSOD lost its stability at pH > 9. The temperatures of the optimum activities were same for both types of enzymes. However, the heat stability of the 6xHis-FeSOD was clearly decreased; even at $20^{\circ}C$ the enzyme lost the activity after 360 min. In contrast, the native FeSOD was stable after 720 min at below $40^{\circ}C$. $H_2O_2$ inhibition was occurred already at 0.5 mM for the 6xHis-tagged enzyme. Therefore, from the results that the 6xHis-FeSOD retained the enzyme activity at pH 6-7 and $20-40^{\circ}C$, it can be assumed that the protein structure became destabilized under different storage conditions and sensitive to the enzyme inhibitor.

• Microbiology and Biotechnology Letters
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• v.23 no.6
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• pp.784-788
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• 1995

The lambda Integrase (Int) carries out site-specific recombination between the two partner DNA sequences, attachment P (attP) and attachment B (attB). In order to study the recombination mechanism, a large quantity of pure integrase is required. Then, we constructed an int gene inserted recombinant plasmid (pNYL3) by using the pQE31 HIS-Tag vector, and produced the fusion protein, 6xHIS-Int from the E. coli TG1 strain carrying the pNYL3 plasmid. The recombinant protein produced was purified by phosphocellulose and Ni$^{++}$-NTA affinity column chromatographies. The result of the in vitro recombination assay using the standard reaction mixture containing 6xHIS-Int and partially purified integration host factor (IHF) showed that the 6xHIS-Int tagged recombination Integrase had the full recombination activity.

• International Journal of Industrial Entomology and Biomaterials
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• v.17 no.1
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• pp.137-141
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• 2008

We previously isolated and cloned a cDNA of abaecin from the Bombus ignitus. In an effort to produce a large amount of soluble abaecin at low cost, we successfully expressed the peptide in Escherichia coli that are highly sensitive to its mature form. For this, we fused the peptide encoding 39 amino acids of mature B. ignitus abaecin to the thioredoxin gene together with a C-terminal 6xHis tag. An enterokinase cleavage site was introduced between the 6xHis tag and mature abaecin to allow final release of the recombinant peptide. A high yield of 9.6 mg soluble fusion protein from 200 ml of bacterial culture was purified by $Ni^{2+}$-charged His-Bind resin affinity column, and 1.4 mg of pure active recombinant abaecin was readily obtained by enterokinase cleavage, followed by affinity chromatograph. The molecular mass of recombinant abaecin peptide was determined by Tricin-SDS-PAGE analysis. The recombinant abaecin exhibited antibacterial activity against Gram-negative bacteria.

• KSBB Journal
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• v.21 no.3
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• pp.204-211
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• 2006

For the production of the recombinant human interferon-gamma(rhIFN-${\gamma}$) in Escherichia coli, human glucagon and ferritin heavy chain were used as fusion partners. Even though rhIFN-${\gamma}$ is expressed as an inclusion body form in E. coli because of strong hydrophobicity of itself, over 50% of fused rhIFN-${\gamma}$ was expressed as soluble form in E. coli $Origami^{TM}$(DE3) harboring pT7FH(HE)-IFN-${\gamma}$ which encodes ferritin heavy chain-fused rhIFN-${\gamma}$. In the case of using glucagon-ferritin heavy chain hybrid mutant as a fusion partner, 6X His-tag was additionally introduced to N-terminus of GFHM(HE)-IFN-${\gamma}$ for enhancing purification yields of rhIFN-${\gamma}$. Fusion protein HGFHM(HE)-IFN-${\gamma}$ with two 6X His-tag was more effectively bound to Ni-NTA agarose bead than GFHM(HE)-IFN-${\gamma}$ with a 6X His-tag. rhIFN-${\gamma}$ was completely purified from enterokinase-treated HGFHM(HE)-IFN-${\gamma}$ by Ni-NTA affinity column. For high-level production of rhIFN-${\gamma}$, glucose was used as the sole carbon source with simple exponential feeding rate($2.4{\sim}7.2g/h$) in fed-batch process. The effective lactose concentration for the expression of the rhIFN-${\gamma}$ was $10{\sim}20mM$. Under the fed-batch culture conditions, rhIFN-${\gamma}$ production yield reached 11 g DCW/L for 6 hours after lactose induction.

• Journal of Life Science
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• v.28 no.1
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• pp.78-82
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• 2018

Detection of pathogenic microorganisms takes several days by conventional methods. It is necessary to assess microorganisms in a timely manner to reduce the risk of spreading infection. For this purpose, bacteriophages are chosen for use as a biosensing tool due to their host specificity, wide abundance, and safety. However, their lytic cycle limits their efficacy as biosensors. Phage proteins involved in binding to bacteria could be a robust alternative in resolving this drawback. Here, a fragment of tail protein J (residues 784 to 1,132) of phage lambda fused with 6X His-tag (6HN-J) at its N-terminus was cloned, overexpressed, purified, and characterized for its binding with microorganisms. The purified protein demonstrated a size of about 38 kDa in sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) and bound with anti-His monoclonal antibodies. It bound specifically to Escherichia coli K-12, and not Salmonella typhimurium, Bacillus subtilis, or Pseudomonas aeruginosa in dot blotting. Binding of the protein to E. coli K-12 inhibited about 50% of the in vivo adsorption of the phage lambda to host cells at a concentration of $1{\mu}g/ml$ 6HN-J protein and almost 100% at $25{\mu}g/ml$ 6HN-J. The results suggest that a fusion viral protein could be utilized as a biosensing element (e.g., protein chips) for detecting microorganisms in real time.

• Microbiology and Biotechnology Letters
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• v.47 no.4
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• pp.536-545
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• 2019

Cytochrome P450 (P450 or CYP) is involved in the metabolism of endogenous and exogenous compounds in most organisms. P450s have great potential as biocatalysts in the pharmaceutical and fine chemical industries because they catalyze diverse oxidative reactions using a wide range of substrates. The high-cost nicotinamide cofactor, NADPH, is essential for P450 reactions. Glucose-6-phosphate dehydrogenase (G6PDH) has been commonly used in NADPH-generating systems (NGSs) to provide NADPH for P450 reactions. Currently, only two G6PDHs from Leuconostoc mesenteroides and Saccharomyces cerevisiae can be obtained commercially. To supply high-cost G6PDH cost-effectively, we cloned the cytosolic G6PDH gene of Solanum lycopersicum (tomato) with 6xHis tag, expressed it in Escherichia coli, and purified the recombinant G6PDH (His-G6PDH) using affinity chromatography. In addition, enzymatic properties of His-G6PDH were investigated, and the His-G6PDH-coupled NGS was optimized for P450 reactions. His-G6PDH supported CYP102A1-catalyzed hydroxylation of omeprazole and testosterone by NADPH generation. This result suggests that tomato His-G6PDH could be a cost-effective enzyme source for NGSs for P450-catalyzed reactions as well as other NADPH-requiring reactions.

• Journal of Dairy Science and Biotechnology
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• v.29 no.1
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• pp.49-54
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• 2011

Bile salt hydrolase (BSH, EC 3.5.1.24) activity, which cleaves amide bond between carboxyl group (bile acid) and amino group (glycine or taurine), is commonly detected in gut-associated species of human and animal. During the screening of BSH active strains from the fecal samples of elderly human volunteers, strain CU30-2 was isolated on the basis of the highly active BSH producing activity. A bsh gene of the isolate was cloned into the pET22b expression vector and overexpressed in Escherichia coli BL21 (DE3) Gold by induction with 1mM IPTG. The overexpressed BSH enzyme with 6x His-tag was purified with apparent homogeneity using a $Ni^+$-NTA agarose column and characterized. The BSH enzyme of E. faecalis CU30-2 exhibited approximately 50 times higher activity against glycol-conjugated bile salts than tauro-conjugated bile salts having the highest activity against glycocholic acid. Considering the prevalence of E. faecalis strains in the human GI tract and glycol-conjugates dominated bile acid composition of human bile, further study is needed to investigate the impact of the BSH activity exerted by E. faecalis strains to the host as well as to the BSH producing strains.

• Korean Journal of Microbiology
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• v.52 no.4
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• pp.495-499
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• 2016

In order to identify the biochemical characteristics of LuxG, the luxG gene from bioluminescence bacteria of Photobacterium leiognathi ATCC 25521 was isolated by PCR-Amplification and inserted into pQE30 vector containing the T5 promoter and 6X His-tag system. The resulting recombinant plasmid was transformed into Escherichia coli to over-express the luxG gene and purify the gene product. The purified LuxG protein demonstrated ferric iron reductase activity and the kinetic parameters of $K_m$ and $V_{max}$ for FMN as well as the NADPH substrates of ferric iron reductase were determined, respectively.

• Molecules and Cells
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• v.25 no.1
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• pp.43-49
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• 2008

Cancer Osaka thyroid, also known as Tpl-2 (Cot) is a member of the MAP3K kinase family and plays a key role in the regulation of the immune response to pro-inflammatory stimuli such as lipopolysaccharide (LPS) and tumour necrosis $factor-{\alpha}$ ($TNF-{\alpha}$). A series of Cot constructs with an N-terminal 6xHis tag were transiently expressed in HEK293 cells: $Cot_{130-399}$ (kinase domain), $Cot_{1-388}$ (N-terminal and kinase do-mains), $Cot_{1-413}$, $Cot_{1-438}$ (containing a putative PEST sequence), $Cot_{1-457}$ (containing both PEST and degron sequences) and $Cot_{1-467}$ (full-length protein). These Cot proteins were pulled down using an anti-6xHis antibody and separated by 2D electrophoresis. The gels were silver-stained and 21 proteins were detected that did not appear, or had substantially reduced intensity, in the control sample. Three of these were identified by MS and MS/MS analysis as Hsp90, Hsp70 and Grp78. Hsp90 appeared to bind to the kinase domain of Cot and this interaction was further investigated using co-immuno-precipitation with both overexpressed Cot in HEK293 cells and endogenous Cot in Hela cells.

• Journal of Life Science
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• v.32 no.12
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• pp.956-961
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• 2022

Plant Chloroplast have several advantages as an expression platform of biopharmaceuticals over conventional expression platforms such as mammalian cells, yeast and bacteria. First, plants do not serve as a host for mammalian infectious virus and have endotoxin like bacteria which can cause anaphylactic shock. In addition, high copy number of chloroplast genome allows for chloroplast transformants to reach the high level of expression of heterologous genes. Moreover, the integration of transgenes into specific region of chloroplast genomes makes chloroplast transformants unaffected by positional effect which can be frequently observed from nuclear transformants, resulting in loss of transgene expressions. Antimicrobial peptides (AMPs) are a kind of innate immunity which is found from bacteria to humans. Unlike conventional antibiotics, very less dosage of AMPs can have catastrophic effect on bacterial survival. Further, the repeated use of AMPs does not trigger the development of bacterial resistance. Moricin, one of the AMPs, was isolated from Bombyx mori, a silkworm moth. The C-terminal of moricin consists largely of basic amino acids, and the N-terminal has an α-helix structure. Moricin was chosen and expressed in a SUMO/SUMOase without leaving any unwanted amino acids which could potentially affect the anti-bacterial activity of the moricin. The transformation vector used in this study has already been created in this lab for the expression in both prokaryotic systems such as E. coli and chloroplast. The expressed moricin was purified using Ni columns and SUMOase, and the antibacterial activity of the purified moricin was confirmed using an agar diffusion assay.