• BMB Reports
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• v.28 no.4
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• pp.353-358
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• 1995

Epitope tagging is the process of fusing a set of amino acid residues that are recognized as an antigenic determinant to a protein of interest. Tagging a protein with an epitope facilitates various immunochemical analyses of the tagged protein with a specific monoclonal antibody. The monoclonal antibody H8 has subtype specificity for an epitope derived from the preS2 region of hepatitis B virus surface antigen. Previous studies on serial deletions of the preS2 region indicated that the preS2 epitope was located in amino acid residues 130~142. To test whether the amino acid sequence in this interval is sufficient to confer on proteins the antigenicity recognizable by the antibody H8, the set of amino acid residues in the interval was tagged to the amino terminal of ${\beta}$-galactosidase and to the carboxyl terminal of the truncated $p56^{lck}$ fragment. The tagged ${\beta}$-galactosidase, expressed in Escherichia coli, maintained the enzymatic activity and was immunoprecipitated efficiently with H8. The tagged $p56^{lck}$ fragment, synthesized in an in vitro translation system, was also immunoprecipitated specifically with H8. These results demonstrate that the amino acid sequence of the preS2 region can be used efficiently for the epitope tagging approach.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.115-121
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• 2011

hFSH is a glycoprotein secreted from anterior pituitary and consists of ${\alpha}$ and ${\beta}$ subunits. Because of its major biological functions including sperm formation in the male and for follicular growth, FSH is used to cure woman's sterility. In this study we tried to produce recombinant hFSH in vitro using a retrovirus expression vector. Two major components of the vector we constructed are: ( i ) a DNA fragment containing ${\alpha}$ and ${\beta}$ genes fused by a DNA sequence coding carboxyl terminal peptide (CTP) of human chorionic gonadotropin, (ii) a DNA fragment corresponding woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Evaluation of expression profile of the recombinant FSH using reverse transcription PCR and enzyme-linked immunosorbent assay (ELISA). Among three cell lines tested, HeLa cells were the best for hFSH expression (5,395 mIU/ml), then followed by chicken embryonic fibroblast (CEF) cells and Chinese hamster ovary (CHO) cells in the order of hFSH production. In addition to the amount, the FSH produced from HeLa cells was highest in terms of biological activity which was determined by measuring cAMP.

• Journal of the Korean Chemical Society
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• v.34 no.3
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• pp.288-296
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• 1990

Antielastic fragment in GBⅠ-Ⅱ differ on $P_1$ site with that of antitryptic fragment in LBI. To obtain further understanding of the role of amino acid residue near the reactive site, specificity of $P_1$ site and loop size, Tyr substituted cyclic nonapeptide and cyclic pentapeptide were synthesized and the inhibition constants for some proteinases were calculated by Dixon method. Cyclic nonapeptide showed no inhibition for chymotrypsin but appeared low inhibitory activity for trypsin and elastase and that of cyclic pentapeptide possessed inhibitory activity for chymotrypsin.

• Bulletin of the Korean Chemical Society
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• v.22 no.1
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• pp.68-76
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• 2001

We have employed chemical modification to identify amino acids essential for the catalytic activity of alliinase (EC 4.4.1.4) from garlic (Allium sativum). Alliinase degrades S-alkyl-L cysteine sulfoxides, causing the characteristic odor of garlic. The activity of alliinase was rapidly and completely inactivated by N-bromosuccinimide(NBS) and slightly decreased by succinic anhydride and N-acetylimidazole. These results indicate that tryptophanyl, lysyl, and tyrosyl residues play an important role in enzyme catalysis. The reaction of alliinase with NBA yielded a characteristic decrease in both the absorbance at 280 nm and the intrinsic fluorescence at 332 nm with increasing reagent concentration of NBS, consistent with the oxidation of tryptophan residues. Kinetic analysis, fluorometric titration of tryptophans and correlation to residual alliinase activity showed that modification of only one residue present on alliinase led to complete inhibition of alliinase activity. To identify this essential tryptophan residue, we employed chemical modification by NBS in the presence and absence of the protecting substrate analogue, S-ethyl-L-cysteine (SEC) and N-terminal sequence analysis of peptide fragment isolated by reverse phase-HPLC. A fragment containing residues 179-188 was isolated. We conclude that Trp182 is essential for alliinase activity.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.6 no.2
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• pp.134-143
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• 2005

Collagen is the important structural proteins in mammals with various peptide composition and cross-linkings. The direct analysis of collagen protein was not suitable because of its structural complexity and diversity. In this study, we suggest the simple way of collagen analysis by introducing matrix-assisted laser desorption/ionization time-of flight mass spectrometry (MALDI-TOF MS) to identify the collagen and its trypsin-digested fragments, and by subsequent time-of-flight tandem mass spectrometry(Q-TOF MS/MS) to analyze the amino acid sequences of identified fragments. Using the collagen samples extracted from the tail of mouse, 10 separated bands were found in SDS-PAGE, and the masses of most bands could be more finely determined by MALDI-TOF MS. When each 10 separated proteins was tryptic digested and introduced to MALDI-TOF, the Gly1056-Arg1073 fragment from $\alpha_1$-chain was identified in four bands, and the Gly1056-Arg1073 fragment from $\alpha_2$-chain was identified in five bands, both in type I collagen. Although few fragments were found because of the cross-linkings left in digested collagen sample, it could be determined that the type I collagen existed at least in 7 separated bands. When the amino acid sequences of two identified fragments were analyzed by Q-TOF MS/MS, both sequences were identical with those determined by MALDI-TOF MS. It suggested that the two peaks in MALDI-TOF MS caused by the fragments identified in this work could be used as the fingerprint to simply identify type I collagen in protein samples.

• Herbal Formula Science
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• v.11 no.2
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• pp.95-110
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• 2003

Alzheimer's disease(AD) is a geriatric dementia that is widespread in old age. In the near future AD will be the biggest problem in public health service. Although a variety of oriental prescriptions in study Jeonmaedan have been traditionally utilized for the treatment of AD, their pharmacological effects and action mechanisms have not yet fully elucidated. It has been widely believed that A${\beta}$ peptide devided from APP causes apoptotic neurotoxicity in AD brain. However, recent evidence suggests that CTl05(carboxy terminal 105 amino acid peptide fragment of APP) may be an important factor causing neurotoxicity in AD. In addition, AD is one of brain degeneration disease. So we studied on herbal medicine that have a relation of brain degeneration. In Oriental Medicine, Jeonmaedan has been used for disease in relation to brain degeneration. As the result of this study, in Jeonmaedan the apoptosis in the nervous system is inhibited, the repair against the degerneration of SK-N-SH cell lines by CT105 expression is promoted. So Jeonmaedan may be beneficial for the treatment of AD.

• BMB Reports
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• v.39 no.1
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• pp.16-21
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• 2006

The coat protein (CP) of Papaya ringspot virus (PRSV) was analyzed for presentation of the antigenic peptide of animal virus, Canine parvovirus (CPV), in Escherichia coli (E. coli). The 45 nucleotides fragment coding for the 15-aa peptide epitope of the CPV-VP2 protein was either inserted into the PRSV-cp gene at the 5', 3' ends, both 5' and 3' ends or substituted into the 3' end of the PRSV cp gene. Each of the chimeric PRSV cp genes was cloned into the pRSET B vector under the control of the T7 promoter and transformed into E. coli. The recombinant coat proteins expressed from different chimeric PRSV-cp genes were purified and intraperitoneally injected into mice. All of the recombinant coat proteins showed strong immunogenicity and stimulate mice immune response. The recombinant coat proteins containing the CPV epitope insertion at the C terminus and at both N and C termini elicited ten times higher specific antisera in immunized mice compared with the other two recombinant coat proteins which contain the CPV epitope insertion at the N terminus and substitution at the C terminus.

• Fisheries and Aquatic Sciences
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• v.18 no.2
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• pp.131-135
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• 2015

Two new bitter-tasting cyclic peptides comprising six amino acids, namely gamakamide C and D, were isolated from cultured oysters Crassostrea gigas. Dimethylaminoazobenzene sulfonyl-amino acid analysis detected Val and Leu in gamakamide C and Ile and Leu in gamakamide D. The molecular formula of gamakamide C was determined as $C_{43}H_{60}N_{7}O_8S$ by high-resolution fast atom bombardment mass spectroscopy (HR FAB-MS) ($[M+H]^+m/z822.4200{\Delta}-2.4mmu$), and that of gamakamide D was determined as $C_{43}H_{62}N_7O_8S$ by HR FAB-MS ($[M+H]^+m/z836.4379{\Delta}-2.0mmu$). Comparison of amino acid analyses and fragment ions by MS/MS among gamakamide C, D, and E (known), the structures of gamakamide C and D were confirmed $as-{\small{L}}-Val-{\small{L}}-Met(SO)-{\small{L}}-NMe-Phe-{\small{L}}-Leu-{\small{D}}-Lys-{\small{L}}-Phe-$ and $-{\small{L}}-Ile-{\small{L}}-Met(SO)-{\small{L}}-NMe-Phe-{\small{L}}-Leu-{\small{D}}-Lys-{\small{L}}-Phe-$, respectively.

• Journal of Microbiology and Biotechnology
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• v.7 no.2
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• pp.132-137
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• 1997

A novel protein (HEAAE, High Essential Amino Acid Encoding Protein), rich in essential amino acids ($75{\%}$ of total), was designed and constructed in our laboratory. The designed peptides were analyzed by SYBLE and stable secondary and tertiary structures were predicted. The monomeric form (HEAAE-1) of the protein consists of 20 amino acid residues with four additional amino acids comprising a potential ${\beta}$-turn (HEAAE-4). Size exclusion analysis demonstrated that the monomer is self-aggregates in aqueous solution to form higher ordered multimeric structures, which are very reminiscent of natural plant storage proteins. The DNA encoding this amino acid sequence was synthesized, and from this monomeric gene fragment (heaae-1), the stable tetrameric form of the gene (heaae-4) was generated by subcloning into the E. coli expression vector pKK223-3. A clear 6 kDa polypeptide band corresponding to the molecular weight of the dimeric form (HEAAE-2) was detected. The smeared band which appeared around the molecular weight corresponding to HEAAE-4 of 11 kDa suggested that the tetramer form of this protein might be processed into smaller size products.

• Mass Spectrometry Letters
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• v.13 no.4
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• pp.177-183
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• 2022

The monomer and dimer structures of the amyloid fragment Aβ(1-16) sequence formed in H₂O were investigated using electrospray ionization mass spectrometry (MS) and tandem MS (MS/MS). Aβ16 monomers and dimers were indicated by signals representing multiple proton adduct forms, [monomer+zH]ⁿ⁺ (=M^z+, z = charge state) and [dimer+zH]^z+ (=D^z+), in the MS spectrum. Fragment ions of monomers and dimers were observed using collision-induced dissociation MS/MS. Peptide bond dissociation was mostly observed in the D1-D7 and V11-K16 regions of the MS/MS spectra for the monomer (or dimer), regardless of the monomer (or dimer) charge state. Both covalent and non-covalent bond dissociation processes were indicated by the MS/MS results for the dimers. During the non-covalent bond dissociation process, the D³⁺ dimer complex was separated into two components: the M¹⁺ and M²⁺ subunits. During the covalent bond dissociation of the D³⁺ dimer complex, the b and y fragment ions attached to the monomer, (M+b_10-15)^z+ and (M+y_9-15)^z+, were thought to originate from the dissociation of the M²⁺ monomer component of the (M¹⁺+M²⁺) complex. Two different D³⁺ complex geometries exist; two distinguished interaction geometries resulting from interactions between the M¹⁺ monomer and two different regions of M²⁺ (the N-terminus and C-terminus) are proposed. Intricate fragmentation patterns were observed in the MS/MS spectrum of the D⁵⁺ complex. The complicated nature of the MS/MS spectrum is attributable to the coexistence of two D⁵⁺ configurations, (M¹⁺+M⁴⁺) and (M²⁺M³⁺), in the Aβ16 solution.