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Yunqi Ma,Ming Lu,Ziwei Chang,이추,유해균,박장수 한국생물공학회 2018 Biotechnology and Bioprocess Engineering Vol.23 No.1
Cytochrome P4501A (CYP1A) has attracted an increasing interest due to its important role in metabolism of contaminants in aquatic environment and kinds of biomarkers to monitor the pollutants. CYPs are reported to express in E. coli, yeast and insect cells, while expression levels in these systems are too low to continue further study, such as functional and structural research. In this study, we construct an expression system using Shewanella oneidensis to produce goldfish CYP1A. RBS sequence that can elevate expression level by initiating the translation was added. A leading signal peptide which will direct the goal protein into periplasm of the host was introduced. Moreover, large-size plasmid construction strategy was applied during the successful construction process of expression system. At the position of ~60 kDa, a single band was seen clearly after expression; furthermore the amount of expressed CYP1A was as high as 0.02 micromoles per liter in the culture. Heme test was also performed, the result showed the typical P450 hemoprotein spectra. All these data suggest the possible suitable expression system for fish P4501A system was constructed.
Peptides Containing Multiple Disulfide-Bond Mosaic Expression in the Periplasm of Escherichia coli
Yunqi Ma,김소선,Dong-Geon Kwag,Seo-Hyun Kim,Seung-Ho Ryu,이동훈,소재형,이추,남명모,박장수 대한화학회 2016 Bulletin of the Korean Chemical Society Vol.37 No.9
Peptides containing multiple disulfide bonds are usually problematic when expressed in Escherichia coli. We conducted the expression of β-defensin with three disulfide bonds, hepcidin with four disulfide bonds, and DkTx with six disulfide bonds using a small leader protein mosaic expression in the periplasm of E. coli, and purified them by affinity chromatography and characterized them by mass spectroscopy. The result showed that the expression level was high. A large amount of the pure recombinant peptide was also recovered after purification with a Ni2+ affinity column. The mass results by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) indicated that the recombinant peptides had a folding structure, with the native status of all of the cysteines participating in disulfide bond formation. Moreover, after removing the tags, the result was identical to its natural form as well. We thus provide a method for producing large amounts of soluble peptides containing multiple disulfide bonds in E. coli.
Chang, Ziwei,Lu, Ming,Ma, Yunqi,Kwag, Dong-Geon,Kim, Seo-Hyun,Park, Ji-Min,Nam, Bo-Hye,Kim, Young-Ok,An, Cheul-Min,Li, Huayue,Jung, Jee H,Park, Jang-Su Springer-Verlag ; Springer 2015 Amino acids Vol.47 No.3
<P>Recombinant expression in Escherichia coli allows the simple, economical, and effective production of bioactive peptides. On the other hand, the production of native peptides, particularly those rich in disulfide bonds, is a major problem. Previous studies have reported that the use of carrier proteins for fusion expression can result in good peptide yields, but few are folded correctly. In this study, two transmembrane small proteins in E. coli, YoaJ and YkgR, which both orientate with their N-termini in cytoplasm and their C-termini in periplasm, were used for fusion expression. The recombinant production of two peptides, asteropsin A (ASPA) and 관-defensin (BD), was induced in the periplasm of E. coli using a selected carrier protein. Both peptides were expressed at high levels, at yields of approximately 5-10 mg/L of culture. Mass spectrometry showed that the resulting peptide had the same molecular weight as their natural forms. After purification, single peaks were observed by reversed phase high-performance liquid chromatography (RP-HPLC), demonstrating the absence of isoforms. Furthermore, cytoplasmically expressed fusion proteins with a carrier at their C-termini did not contain disulfide bonds. This study provides new carrier proteins for fusion expression of disulfide bond-rich peptides in E. coli.</P>
Development of Elisa System Using Vitellogenin for Sex Identification of Geoduck (Panopea japonica)
Kim, So-Sun,Ma, Yunqi,So, Jae-Hyeong,Lee, Dong-Hoon,Maeng, Chang-Hyun,Yoo, Hae-Kyun,Lim, Hyun-Jeong,Nam, Myung-Mo,Sohn, Saebom,Park, Jang-Su BioOne (National Shellfisheries Association) 2018 Journal of shellfish research Vol.37 No.5