• Korean Journal Plant Pathology
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• v.10 no.1
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• pp.1-6
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• 1994

Randomly chosen recombinant clones of Fusarium oxysporum were analysed to select useful probes for relatedness analysis of Fusarium oxysporum. Genomic DNA of F. oxysproum f. sp. cubense, digested with HindIII, was ligated to pUC118 and used to transform Escherichia coli strai DH5$\alpha$. Three clones were identified that hybridized to mutiple restriction fragments of some formae speciales of F. oxysporum. These probes detected repetitive sequences in HindIII or EcoRI digested DNAs. Repeated copy clone pFC46, pFC52 and pFC54 showed evident polymorphisms among ten formae speciales of this fungus. Since clone pFC 52 strongly hybridized to multiple EcoRI-digested restriction fragments of f. sp. cubense, it may be useful as a probe for analysis of other genetic characteristics of this forma specialis. The results suggest that our clones might be very useful as probes for relatedness analysis between or within formae speciales of Fusarium oxysporum.

• Biomolecules & Therapeutics
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• v.4 no.2
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• pp.138-147
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• 1996

DWP401, a recombinant human epidermal growth factor, was subcutaneously administered to ICR mice at the dose levels of 0, 0.04, 0.2 and 1.0 mg/kg/day (15rats/sex/group) in order to evaluate the subchronic toxicity. General observations, examinations for food and water consumption, ophthalmoscopy and urinalysis were carried out during the study. For the complete gross and microscopic examinations, 10 mice/ sex/group were sacrificed at the ends of the dosing period, and the remaining animals were sacrificed with a 5 week recovery period. Examinations for hematology and blood biochemistry were also carried out at the time of recovery period. Based on the results, it was thought that the target tissue or organs were mesothelial cell, injection site, spleen, adrenal gland, ovary and transitional epithelial cell of urinary tract, and no observed toxic level of DWP401 was 0.04 mg/kg while definite toxic dose level might be 0.2 mg/kg.

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.233-233
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• 2005

• ALGAE
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• v.18 no.1
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• pp.95-100
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• 2003

• Bulletin of the Korean Chemical Society
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• v.29 no.12
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• pp.2449-2452
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• 2008

• Korean Chemical Engineering Research
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• v.47 no.5
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• pp.624-629
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• 2009

It is the most important step to screen soluble and insoluble proteins when we attempt to improve the solubility of recombinant proteins through directed evolution approach. Here we show that the solubility of a recombinant protein in vivo can be examined by expressing the recombinant protein with beta-lactamase as a fusion partner. First we constructed an expression system which can produc a fusion protein with the C-terminal of beta-lactamase. Two soluble proteins, i.e. adenine deaminase and aspartate aminotransferase, and insoluble GlcNAc-2-epimerase were cloned into the developed expression vector, respectively. We investigated the effect of the expression of the three recombinant fusion proteins on the growth of E. coli, and confirmed that the solubilities of the recombinant proteins correlated with cell growth rates.

• Journal of Microbiology
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• v.40 no.1
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• pp.77-81
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• 2002

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic hematspoietic growth factor involved in the development of myeloid cells from bone marrow, and an activator of mature myeloid cells functioning in a variety of antimicrobial and inflammatory responses. Recently, recombinant GM-CSF is increasingly under clinical study for treatment of various diseases including cancer, infectious diseases and hematopoietic diseases as well as for an immune response modulator, In this study, we constructed a recombinant human GM-CSF (rhGM-CSF) expression plasmid with a pelB leader sequence and His. Tag under T7 promoter control. The expression construct was shown to produce a recombinant protein of 20 kDa in the 8M urea preparation, indicating the rhGM-CSF may be expressed as an insoluble inclusion form. The 20 kDa recombinant protein in 8M urea was transformed into the water-so1ub1e form by dialysis against PBS buffer (phosphate buffered saline). The soluble rhGM-CSF protein was shown to stimulate colony formation and cell proliferation in vitro, indicating that the rhGM-CSF could be refolded into its native form to show colony stimulating activity.

• BMB Reports
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• v.32 no.5
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• pp.461-467
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• 1999

Recombinant Mycobacterium smegmatis expressing ovalbumin was used to immunize C57BL/6(H-$2^b$) mice, and the humoral immunity against recombinant ovalbumin was analyzed. Antibodies were purified by denatured ovalbumin-conjugated affinity chromatography. The epitopes of the antibodies were screened with a random peptide library displayed on the tip of fUSE5 filamentous phage pIII minor coat proteins. Two peptides, IRLADR and SPGAEV, were selected predominantly by the recognition of purified antibodies using biopanning methods. The composition of the peptide sequence with the primary structure of OVA revealed that the peptide sequence analogizes to INEAGR, part of the $^{323}ISQAVHAAHAEINEAGR^{339}$ sequence previously reported as the antigenic determinant for murine Band also Th cell epitopes (I-$A^d$ binding). Also, the structures of these mimotopes obtained from restrained molecular dynamic computations resulted in the formation of a $\beta$-turn proven to be a secondary structure of the parent peptide within the ovalbumin molecule, enabling us to confirm the structural similarity. This study demonstrates that immunization with recombinant M. smegmatis can generate neutralizing antibodies identical with those induced by the administration of natural antigenic proteins and supports the potential use of mycobacteria as vaccine delivery vehicles.

• Journal of Microbiology and Biotechnology
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• v.23 no.8
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• pp.1116-1122
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• 2013

Cell line development is the most critical and also the most time-consuming step in the production of recombinant therapeutic proteins. In this regard, a variety of vector and cell engineering strategies have been developed for generating high-producing mammalian cells; however, the cell line engineering approach seems to show various results on different recombinant protein producer cells. In order to improve the secretory capacity of a recombinant tissue plasminogen activator (t-PA)-producing Chinese hamster ovary (CHO) cell line, we developed cell line engineering approaches based on the ceramide transfer protein (CERT) and X-box binding protein 1 (XBP1) genes. For this purpose, CERT S132A, a mutant form of CERT that is resistant to phosphorylation, and XBP1s were overexpressed in a recombinant t-PA-producing CHO cell line. Overexpression of CERT S132A increased the specific productivity of t-PA-producing CHO cells up to 35%. In contrast, the heterologous expression of XBP1s did not affect the t-PA expression rate. Our results suggest that CERT-S132A-based secretion engineering could be an effective strategy for enhancing recombinant t-PA production in CHO cells.

• Reproductive and Developmental Biology
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• v.33 no.2
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• pp.71-76
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• 2009

Salivary lipocalin (SAL1) is a member of the lipocalin protein family that has a property to associate with many lipophilic molecules. The importance of SAL1 during pregnancy in pigs has been suggested by our previous study which has shown that SAL1 is expressed in the uterine endometrium in a cell type- and implantation stage-specific manner and secreted into the uterine lumen. However, function of SAL1 in the uterus during pregnancy in pigs is not known. To understand SAL1 function in the uterus during pregnancy, we generated recombinant porcine SAL1 protein in an insect cell line. Porcine SAL1 cDNA was cloned into a baculovirus expression vector using RT-PCR and total RNA from uterine endometrium on day 12 of pregnancy, and the expression vector was used to generate recombinant Bacmid containing the SAL1 gene. The recombinant Bacmid was then transfected Sf9 cell to produce recombinant baculovirus. By infecting Sf9 cell with recombinant baculovirus, we established a SAL1-expressing insect cell expression system. Immunoblot analysis confirmed SAL1 expression in the infected cells. Recombinant SAL1 produced by the Sf9 cell line will be useful for understanding physiological function of SAL1 during pregnancy in pigs.