• Archives of Pharmacal Research
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• 제24권6호
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• pp.601-606
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• 2001

A simple assay method of recombinant human epidermal growth factor (rhEGF) in a pharmaceutical preparation was studied and validated by capillary electrophoresis (CE) using micellar electrokinetic chromatography (MEKC) techniques. Factors affecting the migration behavior and separation performances of the peptide; type of buffers pH, butler concentration, and concentration of sodium dodecyl sulfates (SDS) were investigated to optimize the analytical performance. CE was performed using running buffers 50.0 mM borate (pH 8.5) containing 12.5 mM SDS at 20 $mutextrm{V}$ of the applied voltage. Calibration curves for the rhEGF showed good linearity (r>0.999) over the wide dynamic range from 1.25 to $100{\mu\textrm{g}}/ml$. Sample analysis was performed by using standard addition method to eliminate the matrix effects of dosage vehicle. This method is assumed to be useful for quality control (QC) of various forms of pharmaceutical products of the peptide.

• Journal of Microbiology and Biotechnology
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• 제9권3호
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• pp.303-310
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• 1999

A cost-effective mass production method for a strong antimicrobial peptide, buforin II, which was isolated from the stomach of Bufo bufo gargarizans, has been developed. This method is based on the neutralization of the positive charge of buforin II by fusion with a cysteine-rich acidic peptide (CAP) to avoid any lethal effect on the host. The neutralized fusion peptide was multimerized and expressed in Escherichia coli as tandem repeats to increase the production yield. Multimers of the CAP-buforin II fusion peptide were successfully expressed at high levels in E. coli as inclusion bodies. More than 100mg of pure buforin II was obtained per 11 of E. coli culture after cleaving the multimeric polypeptide with CNBr. The buforin II obtained from the recombinant E. coli had antimicrobial activity identical to that of natural buforin II. The proposed expression system can provide a cost-effective mass production method for both antimicrobial peptides and other host-lethal basic proteins.

• Molecules and Cells
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• 제26권2호
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• pp.140-145
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• 2008

Expression of olive flounder hepcidin I (HepI) fused with truncated OmpA signal peptides ($OmpASP_{tr}$) as directional signals does not produce soluble fusion proteins. However, by inserting amino acid segments (xxx) varying in pI and hydrophobicity/hydrophilicity into a leader sequence containing a truncated OmpASP ($OmpASP_{tr}$) and a factor Xa cleavage site (Xa) [$OmpASP_{tr}{\mid}(xxx){\mid}Xa$], we were able in some cases to express soluble recombinant HepI. Soluble expression of the recombinant protein strongly correlated with (xxx) insertions of high pI and hydrophilicity. Therefore, we modified the $OmpASP_{tr}{\mid}(xxx){\mid}Xa$ sequence by inserting Arg and Lys into (xxx) to increase the hydrophilicity of the signal peptide region. These modifications enhanced the expression of soluble recombinant HepI. Hydropathic profile analysis of the $OmpASP_{tr}{\mid}(xxx){\mid}Xa$ HepI fusion proteins revealed that the transmembrane-like domains derived from the $OmpASP_{tr}{\mid}(xxx){\mid}Xa$ sequence were larger than the internal positively charged domain native to HepI. It should therefore be possible to overcome the obstacle of internal positively charged domains to obtain soluble expression of recombinant proteins by monitoring the hydrophilicity and hydropathic profile of the signal peptide region using a computer program.

• 미생물학회지
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• 제36권3호
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• pp.236-241
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• 2000

효모 Saccharomyces cerevisiae를 숙주 세포로 이용하여 활성형의 항균펩티드를 생산하기 위한 model system으로서 쉬파리 유래 defensin의 합성 유전자를 GAP promoter, mating factor $\alpha$1 (MF$\alpha$1)의 preprosequence 및 GAL7 transcription terminator의 조절하에 있는 재조합 plasmid pGMD18을 제작한 후 Saccharomyces cerevisiae에 형질전환하여 growth inhibtion zone assay를 통해 항균활성을 보유한 재조합 효모를 선별하였다. 재조합 효모의 성장 속도와 defensin의 분비능을 증가시키기 위하여 최적의 배지 조성과 배양조건을 결정하였다. 재조합 효모의 defensin 생산을 위한 최적 배양조건을 조사한 결과 0.4% yeast extract, 유기질소원 2% corn steep liquor, 탄소원 2.5% glucose, 0.05% $C_2CO_3$를 포함한 배지에서 초기 pH3 그리고 배양온도 $28^{\circ}C$의 경우가 세포성장과 defensin 의 항균활성이 가장 우수하였다. 이 조건으로 배양을 수행한 결과 YPD 배지에서 배양한 조건보다 약 2배의 세포 성장과 약 4배의 defenzin 생산을 확인하였다.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.20-21
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• 2003

Insulin-like growth factors (IGFs) are mitogenic peptide hormones that regulate embryonic development, postnatal growth and cellular differentiation in vertebrates IGFs are initially translated as pre-pro-peptides and then proteolytically processed to yield the mature IGFs and E-peptides. Like the C-peptide of pro-insulin, the E-peptides of pro-IGFs are generally believed to possess little or no biological activity other than their potential roles in the biosynthesis of the mature IGFs. Like human IGF-1, previous studies in our laboratory showed that the recombinant trout Ea4-peptide of pro-IGF-1 exhibited a dose-dependent mitegenic activity in cultured BALB/3T3 fibroblasts and other non-oncogenic transformed cells (Tian et al., 1999) We have also shown by in vitro and in vivo studies that Ea4-peptide possessed novel anti-tumor activities (Chen et al., 2002, Kuo and Chen, 2002; Kuo and Chen 2003). Recent results of studies conducted in chorionicallantoic membrane of developing chicken embryos revealed that Ea4-peptide of trout pro-IGF-1 also possesses a dose-dependent antiangiogenic activity. Together these results raised the question whether Ea4-peptide of trout pro-IGF-1 may affect heart and blood vessel development and hematopoiesis in fish embryos. (중략)

• Journal of Dairy Science and Biotechnology
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• 제25권2호
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• pp.17-20
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• 2007

Bovine lactoferricin(LFcin B) is a peptide of 25 amino acids that originated from the N terminus of bovine lactoferrin, and is characterized as having potent antimicrobial activity against bacteria, fungi, protozoa and viruses. But, direct expression of Lfcin B is lethal to Escherichia coli. For the efficient production of Lfcin B in microorganism, we developed an expression system in which the gene for cationic Lfcin B was fused to an anionic peptide gene, and successfully expressed the concatemeric fusion gene in E. coli. The purified recombinant Lfcin B was found to have antimicrobial activity, as chemically synthesized Lfcin B peptide does.

• 한국유가공학회:학술대회논문집
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• 한국유가공기술과힉회 2007년도 추계학술발표대회
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• pp.13-20
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• 2007

Bovine lactoferricin(LFcin B) is a peptide of 25 amino acids that originated from the N terminus of bovine lactoferrin, and is characterized as having potent antimicrobial activity against bacteria, fungi, protozoa and viruses. But, direct expression of Lfcin B is lethal to Escherichia coli. For the efficient production of Lfcin B in E. coli, we developed an expression system in which the gene for cationic Lfcin B was fused to an anionic peptide gene, and successfully expressed the concatemeric fusion gene in E. coli. The purified recombinant Lfcin B was found to have antimicrobial activity, as the native Lfcin B peptide does.

• Reproductive and Developmental Biology
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• 제36권3호
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• pp.193-198
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• 2012

This study was to examine expression of the recombinant full-length adiponectin (recombinant adiponectin) in insect ovarian cell culture system and to characterize structural properties of the recombinant adiponectin secreted in medium. Gene construct encoding the recombinant adiponectin contained N-terminal collagen-like domain (110 Amino Acids, AAs), C-terminal globular domain (137 AAs) and C-terminal peptides for detection with V5 antibody (26 AAs included adaptor peptide) and purification using the 6xHis tag (6 AAs). The approximate molecular weight of the product (monomer) was 35 kDa. Molecular mass species of the expressed recombinant adiponectin were monomer (~35 kDa), dimer (~70 kDa), trimer (~105 kDa) and hexamer (~210 kDa). The major secreted species were the LMW forms, such as monomer, dimer, and trimer. There was MMW of hexamer as minor form. HMW multimers (~300 kDa) were shown as a tracer or not detected on the SDS-PAGE in several experiments (data not shown). The multimer forms in this study were not compatible to those in animal or human serum and adipose tissue by other researcher's study in which the major multimer forms were HMW. By protein denaturing experiments with reducing reagent (${\beta}$-MeOH), anionic detergent (SDS) and heat ($95^{\circ}C$) on the SDS-PAGE, not all adiponectin multimers seemed to have disulfide bond linked structure to form multimers. The recombinant adiponectin which expressed in insect ovarian cell culture system seemed to have the limitation as full physiological regulator for the application to animal and human study.

• 대한수의학회지
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• 제43권1호
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• pp.139-144
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• 2003

A chitinase cDNA named CHT1 was cloned from the hard tick, Haemaphysalis longicornis, and the enzymatic properties of its recombinant proteins were characterized. The CHT1 cDNA encodes 930 amino-acid (aa) residues including a 22 aa putative signal peptide, with the calculated molecular mass of the putative mature protein 104 kDa. The E coli-expressed rCHT1 exhibited weak chitinolytic activity against $4MU-(GlcNAc)_3$. The rCHT1 protein with higher activity was obtained using recombinant Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), which expresses rCHT1 under polyhedrin promoter. These findings suggest that the rCHT1 expressed in baculovirus-mediated Sf 9 cells has a high activity than E coli-expressed rCHT1.

• 한국미생물·생명공학회지
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• 제51권3호
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• pp.243-249
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• 2023

We recently found that the insect-derived antimicrobial peptide CopA3 (LLCIALRKK) directly binds to and inhibits the proteolytic activation of caspases, which play essential roles in apoptotic processes. However, the mechanism of CopA3 binding to caspases remained unknown. Here, using recombinant GST-caspase-3 and -6 proteins, we investigated the mechanism by which CopA3 binds to caspases. We showed that replacement of cysteine in CopA3 with alanine caused a marked loss in its binding activity towards caspase-3 and -6. Exposure to DTT, a reducing agent, also diminished their interaction, suggesting that this cysteine plays an essential role in caspase binding. Experiments using deletion mutants of CopA3 showed that the last N-terminal leucine residue of CopA3 peptide is required for binding of CopA3 to caspases, and that C-terminal lysine and arginine residues also contribute to their interaction. These conclusions are supported by binding experiments employing direct addition of CopA3 deletion mutants to human colonocyte (HT29) extracts containing endogenous caspase-3 and -6 proteins. In summary, binding of CopA3 to caspases is dependent on a cysteine in the intermediate region of the CopA3 peptide and a leucine in the N-terminal region, but that both an arginine and two adjacent lysines in the C-terminal region of CopA3 also contribute. Collectively, these results provide insight into the interaction mechanism and the high selectivity of CopA3 for caspases.