• BMB Reports
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• v.34 no.4
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• pp.293-298
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• 2001

Tryptic activation of the 130-kDa Bacillus thuringiensis Cry4B $\delta$-endotoxin produced protease-resistant products of ca. 47 kDa and ca. 21 kDa. The 21-kDa fragment was identified as the N-terminal five-helix bundle (${\alpha}1-{\alpha}5$,) which is a potential candidate for membrane insertion and pore formation. In this study, we constructed the recombinant clone over-expressing this putative pore-forming (PPF) fragment as inclusion bodies in Escherichia coli. The partially purified inclusions were composed of a 23-kDa protein, which cross-reacted with Cry4B antibodies, and whose N-terminus was identical to that of the 130-kDa protein. Dissimilar to protoxin inclusions, the PPF inclusions were only soluble when the carbonate buffer, pH 9.0, was supplemented with 6 M urea. After renaturation via a stepwise dialysis, the refolded PPF protein appeared to exist as an oligomer and was structurally stable upon trypsin treatment. Unlike the 130kDa protoxin, the refolded protein was able to release entrapped glucose from liposomes, and showed comparable activity to the full-length activated toxin, although it lacks larvicidal activity These results, therefore, support the notion that the PPF fragment that consists of ${\alpha}1-{\alpha}5$ of the activated Cry4B toxin is involved in membrane pore-formation.

• 한국생물공학회:학술대회논문집
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• 2002.04a
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• pp.457-460
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• 2002

In an effort to isolate novel strains expressing a thermostable esterase that hydrolyzed the rac-ketoprofen ethyl ester to ketoprofen in the stereospecific manner, we screened various soils and composts from broad ecological niches in which the activity was expected to be found. Three hundreds of microbial strains were tested to determine their ester-hydrolyzing activity by using an agar plate containing insoluble tributyrin as an indicative substrate, and then further screened by activity on the (R,S)-ketoprofen ethyl ester. Twenty-six strains were screened primarily at high growth and incubation temperature and further compared the ability to ethyl ester-hydrolyzing activity in terms of conversion yield and chiral specificity. Consequently, a strain JYl44 was isolated as a novel strain that produced a (R)-stereospecific esterase with high stability and systematically identified as a Bacillus stearothermophilus JY144. The enzyme indeed stables at a broad range of temperature, upto 65 $^{\circ}C$, and pH ranging from 6.0 to 10.0. The optimal temperature and pH for enzymatic conversion were 50 $^{\circ}C$ and 9.0, respectively. Based on the observations that resulted a poor cell growth, and enzyme expression in wild type strain, we further attempted the gene cloning into a general host Escherichia coli and determined its primary structure, concomitantly resulting a high level expression of the enzyme. The cloned gene had an open reading frame (250 amino acids) with a calculated molecular mass of 27.4 kDa, and its primary structure showed a relative high homology (45-52 %) to the esterases from Streptomyces and Bacillus strains. The recombinant whole cell enzyme could efficiently convert the rac-ketoprofen ethyl ester to (R)-ketoprofen, with optical purity of 99 % and yield of 49 %.

• Journal of Microbiology and Biotechnology
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• v.26 no.12
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• pp.2184-2191
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• 2016

The time-consuming and high-cost preparation of soluble peptide-major histocompatibility complexes (pMHC) currently limits their wide uses in monitoring antigen-specific T cells. The single-chain trimer (SCT) of peptide-${\beta}2m$-MHC class I heavy chain was developed as an alternative strategy, but its gene fusion is hindered in many cases owing to the incompatibility between the multiple restriction enzymes and the restriction endonuclease sites of plasmid vectors. In this study, overlap extension PCR and one-step cloning were adopted to overcome this restriction. The SCT gene of the $OVA_{257-264}$ peptide-$(GS_4)_3-{\beta}2m-(GS_4)_4-H-2K^b$ heavy chain was constructed and inserted into plasmid pET28a by overlap extension PCR and one-step cloning, without the requirement of restriction enzymes. The SCT protein was expressed in Escherichia coli, and then purified and refolded. The resulting $H-2K^b/OVA_{257-264}$ complex showed the correct structural conformation and capability to bind with $OVA_{257-264}$-specific T-cell receptor. The overlap extension PCR and one-step cloning ensure the construction of single-chain MHC class I molecules associated with random epitopes, and will facilitate the preparation of soluble pMHC multimers.

• Journal of Microbiology and Biotechnology
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• v.9 no.3
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• pp.376-380
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• 1999

Constructs, encoding a single-chain variable fragment of a catalytic antibody 4f4f (scFv-4f4f) with glycosidase activity, were made by combining the coding sequences for the heavy and light chain variable domains with a sequence encoding a linker (GGGGS). Using three different plasmid systems, single-chain antibodies were expressed separately in Escherichia coli, demonstrating significant differences in the expression level and amounts in soluble form of the recombinant protein. The protein expression from pET3a-scFv-4f4f was up to 20% of the total soluble proteins and, more importantly, the proteins were mostly found in a soluble form. An SDS-PAGE analysis of the purified single-chain proteins, yielding higher than 5mg from a 1-1 culture, showed a single band corresponding to its molecular weight of 29,100. A preliminary study shows that the expressed scFv-4f4f is catalytically active. The catalytic parameters for the hydrolysis of p-nitrophenyl-$\beta$-D-glucopyranoside by scFv-4f4f are being investigated.

• Journal of Microbiology and Biotechnology
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• v.14 no.1
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• pp.90-96
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• 2004

A synthetic defined medium, fortified with amino acids, was developed for the stable production of the kringle fragments of human apolipoprotein(a) (apo(a)), rhLK68. Using a complex rich medium containing yeast extract and a high-cell-density fed-batch culture, the expression level of rhLK68 reached 17% of the total cellular protein, which corresponded to $5\;g\;l^{-1}$ of the culture. To replace the complex media with chemically defined media, several amino acids that positively affect cell growth and gene expression were chosen by a statistical method. The various combinations of the selected amino acids were tested for its fortifying effect on a minimal defined medium. When glutamine only was added, the overall expression level of rhLK68 reached 93% of the complex rich medium increasing the specific expression level by 22.4% and decreasing the cell growth by 24%. Moreover, the addition of glutamine resulted in a 2-fold increase in the concentration of rhLK68 in the culture broth, compared with the minimal defined medium. The synthetic defined media developed in this study could be generally applied to high-cell-density cultures of the recombinant Escherichia coli BL21(DE3), especially for the production of therapeutic proteins that require a strict quality control of the culture media and fermentation processes.

• Food Science and Biotechnology
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• v.14 no.6
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• pp.860-865
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• 2005

A gene for a putative glucoamylase, stg, of a hyperthermophilic archae on Sulfolobus tokodaii was cloned and expressed in Escherichia coli. The recombinant glucoamylase (STGA) had an optimal temperature of $80^{\circ}C$ and was extremely thermostable with a D-value of 17 hr. The pH optimum of the enzyme was 4.5. Being different from fungal glucoamylases, STGA hydrolyzed maltotriose (G3) most efficiently. Gel permeation chromatography and sedimentation equilibrium analytical ultracentrifugation analysis showed that the enzyme existed as a dimer. STGA was stable enough to hydrolyze liquefied com starch to glucose in 4 hr at $90^{\circ}C$ with a yield of95%. Comparison of the $k_{cat}$ values for the hydrolysis and the reverse reaction at $75^{\circ}C$ and $90^{\circ}C$ indicated that glucose production by STGA was more efficient at $90^{\circ}C$ than $75^{\circ}C$. Therefore, STGA showed great potential for application to the industrial glucose production process due to its high thermostability.

• Biomolecules & Therapeutics
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• v.16 no.4
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• pp.336-342
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• 2008

The resveratrol analogue piceatannol (3,5,3',4'-tetrahydroxy-trans-stilbene) is a polyphenol present in grapes and wine and reported to have anti-carcinogenic activities. To investigate the mechanism of anticarcinogenic activities of piceatannol, the effects on CYP 1 enzymes were determined in Escherichia coli membranes coexpressing recombinant human CYP1A1, CYP1A2 or CYP1B1 with human NADPH-P450 reductase. Piceatannol showed a strong inhibition of CYP1A1 and CYP1B1 in a concentration-dependent manner, and $IC_{50}$ of human CYP1A1 and CYP1B1 was 5.8 ${\mu}M$ and 16.6 ${\mu}M$, respectively. However, piceatannol did not inhibit CYP1A2 activity in the concentration of up to 100 ${\mu}M$. Piceatannol exhibited 3-fold selectivity for CYP1B1 over CYP1A1. The mode of inhibition of piceatannol was non-competitive for CYP1A1 and CYP1B1. The result that piceatannol did not inhibit CYP1B1-mediated $\alpha$-naphthoflavone ($\alpha$-NF) metabolism suggests piceatannol may act as a non-competitive inhibitor as well. In human prostate carcinoma PC-3 cells, piceatannol induces apoptosis and prevents Aktmediated signal pathway. Taken together, abilities of piceatannol to induce apoptotic cell death as well as CYP1 enzyme inhibition make this compound a useful tool for cancer chemoprevention.

• Korean Journal of Environmental Biology
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• v.37 no.4
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• pp.585-591
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• 2019

Most insect-resistant LMOs have been produced by applying Cry and Vip3Aa proteins. Vip3Aa protein is activated during the vegetative stage of Bacillus thuringensis (Bt) and the inhibitory activity of the Vip3Aa protein against pathogenic attacks from lepidopteran insect species is well known. However, a risk assessment of the Vip3Aa protein compared to the Cry protein has not been conducted in South Korea. This study demonstrates a possible risk assessment method for Vip3Aa protein against honeybees (Apis mellifera). For the risk assessment of the protein, we purified the recombinant Vip3Aa protein in Escherichia coli. The survival rate and symptoms of general intoxication of 4 months honeybees were measured after Vip3Aa exposure. These results indicated that there was no significant difference in the survival rate and the symptom between Vip3Aa and the control buffer. In this study, we established standard methods of Vip3Aa protein purification and oral adult toxicity test using A. mellifera as an LMO risk assessment technique for preserving the natural ecosystem of South Korea.

• Korean Journal of Veterinary Research
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• v.46 no.2
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• pp.127-133
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• 2006

Pasteurella multocida is a terrible veterinary pathogen that causes widespread infections in husbandry. To induce homologous and/or heterologous immunity against the infections, outer membrane protein Hs (OmpH) in the envelope of different strains of P. multocida are thought to be attractive vaccine candidates. Previously we cloned and characterized a gene for OmpH from pathogenic P. multocida (A : 3) (In Press, Korean J. Microbiol. Biotechnol. 2005, 33, December). The gene is composed of 1,047 nucleotides (nt) coding 348 amino acids (aa) with signal peptide of 20 aa. The truncated ompH, a gene without nt coding for the signal peptide, was generated using pRSET A to name "pRSET A/OmpH-F2". This truncated ompH was well expressed in Escherichia coli BL21 (DE3). Truncated OmpH was purified for induction of immunity against live pathogen of fowl cholera (P. multocida A : 3) in mice. Some $50{\mu}g$ of the purified polypeptide was intraperitoneally injected into mice two times with 10 day interval. Lethal dose ($25{\mu}l$) of live P. multocida A : 3 was determined by directly injecting the pathogen into wild mice (n = 25). To demonstrate the vaccine candidate of the truncated OmpH, the live pathogen ($25{\mu}l$) was challenged with the OmpH-immunized mouse group as well as positive & negative controls (n = 80). The results show that the truncated OmpH can be used for an effective vaccine production to prevent fowl cholera caused by pathogenic P. multocida (A : 3).

• Molecules and Cells
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• v.19 no.3
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• pp.398-401
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• 2005

The bacterial enzyme UDP-N-acetylglucosamine enolpyruvyl transferase catalyzes the first committed step of peptidoglycan biosynthesis, i.e., transfer of enolpyruvate from phosphoenolpyruvate to UDP-N-acetyl-glucosamine. We have overexpressed the enzyme from Haemophilus influenzae in Escherichia coli and crystallized it in the apo-form, as well as in a complex with UDP-N-acetylglucosamine and fosfomycin using ammonium sulfate as the precipitant. X-ray diffraction data from a crystal of the apo-form were collected to $2.8{\AA}$ resolution at 293 K. The crystal quality was improved by co-crystallization with UDP-N-acetylglucosamine and fosfomycin. X-ray data to $2.2{\AA}$ have been collected at 100 K from a flash-frozen crystal of the complex. The complex crystals belong to the orthorhombic space group I222 (or $I2_12_12_1$) with unit-cell parameters of a = 63.7, b = 124.5, and $c=126.3{\AA}$. Assuming a monomer of the recombinant enzyme in the crystallographic asymmetric unit, the calculated Matthews parameter ($V_M$) is $2.71{\AA}^3Da^{-1}$ and solvent content is 54.6%.